CA2420932A1 - Novel mammalian nuclear receptor l66 and methods of use - Google Patents

Abstract

The present invention relates to a novel nuclear receptor called "L66" or also FXR-.beta. a homologue of the FXR-.alpha., a prototypical type 2 nuclear receptor. The invention also relates to the isolated nucleic acid sequence of L66 and the isolated protein thereof. The invention further relates to processes for isolating and/or producing the nucleic acid or the protein as well as methods of use of the receptor L66.

Description

Title:

BACKGROUND OF THE INVENTION
Multicellular organisms are dependent on advanced mechanisms of information transfer between cells and body compartments. The information that is transmitted can be highly complex and can result in the alteration of genetic programs involved in cellular differentiation, proliferation, or reproduction. The signals, or hormones, are often simple molecules, such as peptides, fatty acid, or cholesterol derivatives.
Many of these signals produce their effects by ultimately changing the transcription of specific genes. One well-studied group of proteins that mediate a cell's response to a variety of signals is the family of transcription factors lenown as nuclear receptors, hereinafter referred to often as "NR". Members of this group include receptors for steroid hormones, vitamin D, ecdysone, cis and trans retinoic acid, thyroid hormone, bile acids, cholesterol-derivatives, fatty acids (and other peroxisomal proliferators), as well as so-called orphan receptors, proteins that are structurally similar to other members of this group, but for which no ligands are known (Escriva, H. et al., Ligand binding was acquired during evolution of nuclear receptors, PNAS, 94, 6803 -6808, 1997). Orphan receptors may be indicative of unknown signaling pathways in the cell or may be nuclear receptors that function without ligand activation. The activation of transcription by some of these orphan receptors may occur in the absence of an exogenous ligand and/or through signal transduction pathways originating from the cell surface (Mangelsdorf, D. J. et al., The nuclear receptor superfamily: the second decade, Cell 83, 835-839, 1995).
In general, three functional domains have been defined in NRs. An amino terminal domain is believed to have some regulatory function. A DNA-binding domain hereinafter referred to as "DBD" usually comprises two zinc finger elements and recognizes a specific Hormone Responsive Element hereinafter referred to as "HRE"
within the promoters of responsive genes. Specific amino acid residues in the "DBD"
have been shown to confer DNA sequence binding specificity (Schena, M. &
Yamamoto, K.R., Mammalian Glucocorticoid Receptor Derivatives Enhance Transcription in Yeast, Science, 241:965-967, 1988). A Ligand-binding-domain hereinafter referred to as "LBD" is at the carboxy-terminal region of known NRs. In °:
the absence of hormone, the LBD appears to interfere with the interaction of the DBD
with its HRE. Hormone binding seems to result in a conformational change in the NR
and thus opens this interference (Brzozowski et al., Molecular basis of agonism and antagonism in the oestogen receptor, Nature, 389, 753 - 758, 1997; Wagner et al., A
structural role for hormone in the fihyroid hormone receptor, Nature, 378, 690 - 697.
1995). A NR without the HBD constitutively activates transcription but at a low level.
Both the amino-terminal domain and the LBD appear to have firanscription activation functions hereinafter referred to as "TAF". Acidic residues in the amino-terminal domains of some nuclear receptors may be important for these transcription factors to interact with RNA polymerase. TAF activity may be dependent on interactions with other protein factors or nuclear components (Diamond et al., Transcription Factor Interactions: Selectors of Positive or Negative Regulation from a Single DNA
Element, Science, 249:1266-1272 , 1990). Certain oncoproteins (e.g., c-Jun and c-Fos) can show synergistic or antagonistic activity with glucocorticoid receptors (GR) in transfected cells. Furthermore, the receptors for estrogen and vitamins A
and D, and fatty acids have been shown to interact, either physically or functionally, with the Jun and Fos components of AP-1 in the transactivation of steroid- or AP-1 regulated genes.
Coactivators or transcriptional activators are proposed to bridge between sequence specific transcription factors, the basal transcription machinery and in addition to influence the chromatin structure of a target cell. Several proteins like SRC-1, ACTR, and Grip1 interact with NRs in a ligand enhanced manner (Heery et al., A
signature motif in transcriptional coactivators mediates binding to nuclear receptors, Nature, 387, 733 - 736; Heinzel et al., A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression, Nature 387, 43 - 47, 1997).
Furthermore, the physical interaction with negative receptor-interacting proteins or corepressors has been demonstrated (Xu et al., Coactivator and Corepressor complexes in nuclear receptor function, Curr Opin Genet Dev, 9 (2), 140 - 147, 1999).
Nuclear receptor modulators like steroid hormones affect the growth and function of specific cells by binding to intracellular receptors and forming nuclear receptor-ligand complexes. Nuclear receptor-hormone complexes then interact with a hormone response element (HRE) in, the control region of specific genes and alter specific gene expression.
Over the past decade, new members of the nuclear hormone gene family have been identified that lack known ligands. These orphan receptors can be used to uncover signaling molecules that regulate yet unidentified physiological networks.
Some of these orphan receptors are constitutively active in transactivate target genes without the need to interact with a ligand (MangelsdorFet al., 1995).
Farnesoid X Receptor alpha (hereinafter FXR-a) is a prototypical type 2 nuclear receptor (US Pat. 6,005,086) which activates genes upon binding to promoter region of target genes in a heterodimeric fashion with Retinoid X Receptor (hereinafter RXR, Forman et al., Cell, 81, 687-93, 1995).. The relevant physiological ligands of FXR-a seem to be bile acids. The most potent is chenodeoxycholic acid, which regulates the expression of several genes that participate in bile acid homeostisis. Farnesoid, originally described fio activate the rat ortholog at high concentration does not activate the human or mouse receptor. It is highly expressed in the liver, intestine and kidney. Like LXR-a FXR-a is involved in intracrine signaling.
The relevant physiological ligands of NR1 H4 (as FXR-a is also called) seem to be bile acids (Makishima et al., Science, 284, 1362-65, 1999; Parks et al., Science, 284, 1365-68, 1999). The most potent is chenodeoxycholic acid, which regulates the expression of several genes that participate in bile acid homeostasis.
Consequently, FXR-a is proposed to be a nuclear bile acid sensor. As a result, it modulates both, the synthetic output of bile acids in the liver and their recycling in the intestine (by regulating bile acid binding proteins?. Its is also activated by retoinic acid and TTNPB at supraphisiological concentration. Furthermore, it regulates the conversion of dietry cholesterol into bile acids by regulation the metabolizing genes like CYP7-a. This is a feed back regulation since the receptor is activated by bile acids.
Through its regulatory function in cholesterol and bile acid metabolism an FXR-a homologue could serve as a target for cholesterol lowering drugs and exert beneficial effects in diseases like artheriosclerosis and other metabolic disorders.
It was thus an object of the present invention to provide for a novel nuclear receptor.
In a preferred embodiment of the invention it was an object to provide for a homologue of FXR-a. It was an object of the present invention to provide for means of producing this receptor as well as means of screening for agonists and antagonists to the receptor. Further objects of the invention are outlined below.
SUMMARY OF THE INVENTION
The present invention provides, inter alia, a novel nuclear receptor protein.
In a preferred embodiment of the invention a novel FXR-a homologue is provided for.
Also provided is the nucleic acid sequence encoding this novel nuclear receptor protein, as well as compounds and methods for using this protein and its nucleic acid sequence.

The present invention provides a novel proteins, nucleic acids, and methods useful for developing and identifying compounds for the treatment of such diseases and disorders as metabolic disorders, immunological indications, hormonal dysfunctions, neurosystemic diseases and in preferred embodiments, high cholesterol and artheriosclerosis as well as other metabolic disorders.
Identified and disclosed herein is the protein sequence for a novel nuclear receptor and the nucleic acid sequence encoding this nuclear receptor L66, which we call the L66 nuclear receptor (or simply "L66") receptor, or also FXR-(i.
The importance of this discovery is manifested in the effects of FXR-~3 to modulate genes involved in cellular functions like regulation of metabolism and cell homeostasis, cell proliferation and differentiation, pathological cellular aberrations, or cellular defense mechanisms including tumor development, i.e. cancer.
Thus, this L66 protein is useful for screening for L66 agonists and antagonist activity for controlling these conditions.
In one aspect of the present invention, we provide isolated nucleic acid sequences for a novel receptor, the L66 receptor. In particular, we provide the cDNA
sequences, protein sequences as well as the genomic sequences encoding the human L66 receptor, as well as the cDNA sequence, protein sequence and genomic sequence of the Mus musculus (mouse) receptor.
These nucleic acid sequences have a variety of uses. For example, they are useful for making vectors and for transforming cells, both of which are ultimately useful for production of the L66 protein.
They are also useful as scientific research tools for developing nucleic acid probes for determining L66 expression levels, e.g., to identify diseased or otherwise abnormal states. They are useful for developing analytical tools such as anti sense oligonucleotides for selectively inhibiting expression of the L66 gene to determine physiological responses.

In another aspect of the present invention, we provide a homogenous composition comprising the L66 protein. The protein is useful for screening drugs for agonist and antagonist activity, and, therefore, for screening for drugs useful in regulating physiological responses associated with L66. Specifically, antagonists to the receptor could be used to treat diseases and disorders as metabolic disorders, immunological indications, hormonal dysfunctions, neurosystemic diseases and in preferred embodiments, high cholesterol and artheriosclerosis as well as other metabolic disorders, whereas agonists could be used for the treatment of these conditions. The proteins are also useful for developing antibodies for detection of the protein.
Flowing from the foregoing are a number of other aspects of the invention, including (a) vectors, such as plasmids, comprising the L66 nuclear receptor nucleic acid sequence that may further comprise additional regulatory elements, e.g., promoters, (b) transformed cells that express the L66, (c) nucleic acid probes, (d) antisense oligonucleotides, (e) agonists, (f) antagonists, and (g) transgenic mammals.
Further aspects of the invention comprise methods for making and using the foregoing compounds and compositions.
The foregoing merely summarizes certain aspects of the present invention and is not intended, nor should it be construed, to limit the invention in any manner.
All patents and other publications recited herein are hereby incorporated by reference in their entirety.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
THE L66 PROTEIN AND NUCLEIC ACID:
The present invention comprises a novel member of the nuclear receptor superfamily which the inventors herein refer to as L66. Particularly preferred embodiments of the L66 receptor are those having an amino acid sequence substantially the same as SEQ ID NO. 3, 24 and/or 19. Examination of the amino acid sequence confirms that the present protein is indeed a member of the nuclear receptor family (see also US
6,005,086) which is closely related to FXR (see also figure 3 for the domain composition). The carboxy-terminal ligand binding domain "LBD" of L66 is a complex region encoding subdomains for ligand binding, often dimerization and transcriptional activation.
The nucleic acids claimed above may be present in various forms, i.e. as an RNA, DNA, cDNA or as genomic DNA.
As used herein, if reference to L66, the L66 receptor, the nuclear receptor L66 or the L66 nuclear receptor is made it is meant as a reference to any protein having an amino acid sequence substantially the same as SEQ ID NO.: 3, 24 and/or 19.
The present invention also comprises nucleic acid sequences encoding the L66 receptor, which nucleic acid sequences are substantially the same as SEQ ID
NO.
1 and SEQ ID NO. 17 (cDNAs) or splice variants thereof 4, 6, 8, 10, 12, 22, their sequence complements SEQ ID NO. 2 and 18 or complements of said splice variants 5, 7, 9, 11, 13, 23. SEQ ID NO 4encodes the human cDNA L66 receptor and is a preferred embodiment. SEQ OD NO. 17 (cDNA) encodes the mouse receptor (Mus musculus). SEQ ID NO. 20 represents the genomic sequence of L66 locus from the mouse (Mus musculus) (see also Fig. 15)1n one embodiment, the nucleic acid sequence according to the invention comprises the sequence according to SEQ ID
NO. 1, 4, 6, 8, 10, 12, 17, 20( 4 to 12 representing various splice variants of the human L66) and/or 22 or portions thereof, in a preferred embodiment nucleic acid sequence according to the invention consists of the sequence according to SEQ
ID
NO. 1, 4, 6, 8, 10 ( 4 to 10 representing various splice variants of the human L66) and/or 17 or portions thereof.
Herein the "complement" refers to the complementary strand of the nucleic acid according to the invention, thus the strand that would hybridize to the nucleic acid according to the invention. in accordance with standard biological terminology all DNA sequences herein are however written in 5'-3' orientation, thus the if a complement is mentioned (see also figures) it is actually a "reverse"
complement (as also stated in the figures). For simplification purposes they may however sometimes be referred to simply as "complements".

As used herein, a protein "having an amino acid sequence substantially the same or similar as SEQ ID NO x" (where "x" is the number of one of the protein sequences recited in the Sequence Listing) means a profiein whose amino acid sequence is the same as SEQ ID NO x or differs only in a way such that at least 50% of the residues compared in a sequence alignment with SEQ ID NO. x are identical, preferably 75%
of the residues are identical, even more preferably 95% of the residues are identical and most preferably at least 98% of the residues are identical.
Those skilled in the art will appreciate that conservative substitutions of amino acids can be made without significantly diminishing the protein's affinity for interacting proteins, DNA binding sites, L66 receptor modulators, e.g. small molecular hydrophobic compounds, or RNA.
Other substitutions may be made that increase the protein's affinity for these compounds. Making and identifying such proteins is a routine matter given the teachings herein, and can be accomplished, for example, by altering the nucleic acid sequence encoding the protein (as disclosed herein), inserting it into a vector, transforming a cell, expressing the nucleic acid sequence, and measuring the binding affinity of the resulting protein, all as taught herein.
As used herein the term "a molecule having a nucleotide sequence substantially the same as SEQ ID NO y" (wherein "y" is the number of one of the protein-encoding nucleotide sequences listed in the Sequence Listing) means a nucleic acid encoding a protein "having an amino acid sequence substantially the same as SEQ ID NO
y+I"
(wherein "y+I" is the number of the amino acid sequence for which nucleotide sequence "y" codes) as defined above. This definition is intended to encompass natural allelic variations in the L66 sequence. Cloned nucleic acid provided by the present invention may encode L66 protein of any species of origin, including (but not limited to), for example, mouse, rat, rabbit, cat, dog, primate, and human.
Preferably, the nucleic acid provided by the invention encodes L66 receptors of mammalian, preferably mouse and most preferably human origin.

_ 9 _ Preferably, the L66 receptors proteins provided by the invention are of mammalian, more preferably mouse and most preferably human origin.
The inventors have found (see figures and examples) that the L66 gene in humans is expressed primarily in testis. In mice however expression may be observed also in other tissues.
IDENTIFICATION OF VARIANTS AND HOMOLOGUES AS WELL AS USE OF
PROBES:
Nucleic acid hybridization probes provided by the invention are nucleic acids consisting essentially of the nucleotide sequences complementary to any sequence depicted in SEQ ID NO. 1, 4, 6, 8, 10, 12, 17, 20, 22, 2, 5, 7, 9, 11, 13, 18, 21 and/or 23 or a part thereof and that are effective in nucleic acid hybridization Nucleic acid hybridization probes provided by the invention are nucleic acids capable of detecting i.e. hybridizing to the gene encoding the polypeptides according to SEQ
ID NO. 3, 24 and/or 19.
Nucleic acid probes are useful for detecting L66 gene expression in cells and tissues using techniques well-known in the art, including, but not limited to, Northern blot hybridization, in situ hybridization, and Southern hybridization to reverse transcriptase - polymerase chain reaction product DNAs. The probes provided by the present invention, including oligonucleotide probes derived therefrom, are also useful for Southern hybridization of mammalian, preferably human, genomic DNA for screening for restriction fragment length polymorphism (RFLP) associated with certain genetic disorders. As used herein, the term complementary means a nucleic acid having a sequence that is sufficiently complementary in the Watson-Crick sense to a target nucleic acid to bind to the target under physiological conditions or experimental conditions those skilled in the art routinely use when employing probes.
It is understood in the art that a nucleic acid sequence will hybridize with a complementary nucleic acid sequence under high stringent conditions as defined herein, even though some mismatches maybe present. Such closely matched, but not perfectly complementary sequences are also encompassed by the present invention. For example, differences may occur through genetic code degeneracy, or by naturally occurring or man made mutations and such mismatched sequences would still be encompassed by the present claimed invention.
Preferably, the nucleotide sequence of the nuclear receptor L66 (SEQ ID NO. 1, 4, 17, and/or 22 or splice variants thereof) and/or their complements can be used to derive oligonucleotide fragments (probes) of various length. If the probe is used to detect a human L66 sequence most preferably a complement of SEQ ID NO. 1 and/or 4 or its complement is used. If the probe is supposed to detect a mouse or a rodent sequence probes complementary to SEQ ID NO. 17 and/or 22, or their respective complements are preferred. Stretches of 17 to 30 nucleotides are used frequently but depending on the screening parameters longer sequences as 40, 50, 100, 150 up to the full length of the sequence may be used. Those probes can be synthesized chemically and are obtained readily from commercial oligonucleotide providers. Chemical synthesis has improved over the years and chemical synthesis of oligonucleotides as long as 100-200 bases is possible. The field might advance further to allow chemical synthesis of even longer fragments. Alternatively, probes can also be obtained by biochemical de novo synthesis of single stranded DNA.
In this case the nucleotide sequence of the nuclear receptor L66 or its complement (see figures) serve as a template and the corresponding complementary strand is synthesized. A variety of standard fiechniques such as nick translation or primer extension from specific primers or short random oligonucleotides can be used to synthesize the probe (Molecular Cloning: A Laboratory Manual (3 Volume Set) by Joseph Sambrook, David W. Russell, Joe Sambrook, 2100 pages 3rd edition (January 15, 2001; . Molecular cloning: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, 1989)). Nucleic acid reproduction technologies exemplified by the polymerise chain reaction (Saiki, R.K. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerise. Science 239, 487-491 (1988)) are commonly applied to synthesize probes. In the case of techniques using specific primers the nucleic acid sequence of the nuclear receptor L66 or its complement is not only used as a template in the biochemical reaction but also to derive the specific primers which are needed to prime the reaction.

In some cases one might also consider to use the nucleic acid sequence of the cofactor or its complement as a template to synthesize an RNA probe. A
promoter sequence for a DNA-dependent RNA polymerase has to be introduced at the 5'-end of sequence. As an example this can be done by cloning the sequence infio a vector which carries the respective promoter sequence. It is also possible to introduce the needed sequence by synthesizing a primer with the needed promoter in the form of a 5' "tail". The chemical synthesis of a RNA probe is another option.
Appropriate means are available to detect the event of a hybridization. There is a wide variety of labels and detection systems, e.g. radioactive isotopes, fluorescent, or chemiluminescent molecules which can be linked to the probe. Furthermore, there are methods of introducing haptens which can be detected by antibodies or other ligands such as the avidin/biotin high affinity binding system.
Hybridization can take place in solution or on solid phase or in combinations of the two, e.g. hybridization in solution and subsequent capture of the hybridization product onto a solid phase by immobilized antibodies or by ligand coated magnetic beads.
Hybridization probes act by forming selectively duplex molecules with complementary stretches of a sequence of a gene or a cDNA. The selectivity of the process can be controlled by varying the conditions of hybridization. To select sequences which are identical highly homologous to the sequence of interest stringent conditions for the hybridization will be used, e.g. low salt in the range of 0.02 M to 0.15 M
salt and/or high temperatures in the range from 50°C degrees centigrade to 70°C degrees centigrade. Stringency can be further improved by the addition of formamide to the hybridization solution. The use of stringent conditions which means that only little mismatch or a complete match will lead to a hybridization product would be used to isolate closely related members of the same gene family. Thus, as used herein stringent hybridization conditions are those where between 0.02 M to 0.15 M
salt and/or high temperatures in the range from 50°C degrees centigrade to 70°C
degrees centigrade are applied.

12_ The use of highly stringent conditions or conditions of "high stringency"
means that only very little mismatch or a complete match which lead to a hybridization product would be used to isolate very closely related members of the same gene family.
Thus, as used herein highly stringent hybridization conditions are those where between 0.02 - 0.3 M salt and 65°C degrees centigrade are applied for about 5 to 18 hours of hybridization time and additionally, the sample filters are washed twice for about 15 minutes each at between 60°C - 65°C degrees centigrade, wherein the first washing fluid contains about 0.1 M salt (NaCI and/or Sodium Citrate) and the second contains only about 0.02 M salfi (NaCI and/,or Sodium Citrate). In a preferred embodiment the following conditions are considered to be highly stringent:
Hybridisation in a buffer containing 2 x SSC (0.03 M Sodium Citrate, 0.3 M
NaCI) at 65°C - 68°C degrees centigrade for 12 hours, followed by a washing step for 15 minutes in 0.5 x SSC, 0.1 % SDS, and a washing step for 15 minutes at 65°C degrees centigrade in 0.1 x SSC, 0.1 % SDS.
Less stringent hybridization conditions, e.g. 0.15 M salt - 1 M salt and/or temperatures from 22°C degrees centigrade to 56°C degrees centigrade are applied in order to detect functionally equivalent genes in the same species or for orthologous sequences from other species.
Unspecific hybridization products are removed by washing the reaction products repeatedly in 2 x SSC solution and increasing the temperature.
DEGENERATE PCR AND CLONING OF HOMOLOGUES
The nucleotide sequence of the nuclear receptor L66 or its complement can be used to design primers for a polymerase chain reaction. Due to the degeneracy of the genetic code the respective amino acid sequence is used to design oligonucleotides in which varying bases coding for the same amino acid are included. Numerous design rules for degenerate primers have been published (Compton et al, 1990).
As in hybridization there are a number of factors known to vary the stringency of the PCR. The most important parameter is the annealing temperature. To allow annealing of primers with imperFect matches annealing temperatures are often much lower than the standard annealing temperature of 55°C, e.g. 35°C
to 52°C degrees can be chosen. PCR reaction products can be cloned. Either the PCR product is cloned directly, with reagents and protocols from commercial manufacturers (e.g.
from Invitrogen, San Diego, USA). Alternatively, restriction sites can be introduced intro the PCR product via a 5'-tail of the PCR primers and used for cloning.
Primers for the amplification of the entire or partial pieces of the L66 gene or mRNA, or for reverse transcription may be designed making use of the sequences according to the invention, i.e. those depicted in the figures' below.
GENETIC VARIANTS
Fragments from the nucleotide sequence of the nuclear receptor L66 (SEQ ID NO.
1, 4 or the mouse, ,i.e. 17 or 22) or their complements can be used to cover the whole sequence with overlapping sets of PCR primers. Also the genomic sequences may be used (see figures for sequences). These primers are used to produce PCR
products using genomic DNA from a human diversity panel of healthy individuals or genomic DNA from individuals which are phenotypically conspicuous. Also the genomic sequences may be used, i.e. that of the human clone as deposited by the applicant (deposit number DSM 14483 ) or that of the mouse according to SEQ ID
NO. 20 (or the complement thereof). The I?CR products can be screened for polymorphisms, for example by denaturing gradient gel electrophoresis, binding to proteins detecting mismatches or cleaving heteroduplices or by denaturing high-performance liquid chromatography. Products which display mutations need to be sequenced to identify the nature of the mutation. Alternatively, PCR products can be sequenced directly opnitting the mutation screening step to identify genetic polymorphisms. If genetic variants are identified and are associated wifih a discrete phenotype, these genetic variations can be included in diagnostic assays. The normal variation of the human population is of interest in designing screening assays as some variants might interact better or worse with a respective lead substance (a pharmacodynamic application). Polymorphisms or mutations which can be correlated to phenotypic outcome are a tool to extend the knowledge and the commercial applicability of the nucleotide sequence of the nuclear receptor L66 or its complement or their gene product, as variants might have a slightly different molecular behavior or desired properties. Disease-causing mutations or polymorphisms allow the replacement of this disease inducing gene copy with a wild-type copy by means of gene therapy approaches and/or the modulation of the activity of the gene product by drugs.
PREPARATION OF POLYNUCLEOTIDES:
DNA which encodes receptor L66 may be obtained, in view of the instant disclosure, by chemical synthesis, by screening reverse transcripts of mRNA from appropriate cells or cell line cultures, by screening genomic libraries from appropriate cells, or by combinations of fihese procedures, as illustrated below.
Screening of mRNA or genomic DNA may be carried out with oligonucleotide probes generated from the L66 nucleotide sequences information provided herein.
Probes may be labeled with a detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with known procedures and used in conventional hybridization assays, as described in greater detail in the Examples below. Alternatively, the L66 nucleotide sequence may be obtained by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers being produced from the L66 nucleotide sequences provided herein.
Upon purification or synthesis, the nucleic acid according to the invention may be labeled, e.g. for use as a probe.
As single and differential labeling agents and methods, any agents and methods which are known in the art can be used. For example, single and differential labels may consist of fihe group comprising enzymes such as ~i-galactosidase, alkaline phosphatase and peroxidase, enzyme substrates, coenzymes, dyes, chromophores, fluorescent, chemiluminescent and bioluminescent labels such as FITC, CyS, Cy5.5, Cy7, Texas-Red and IRD40(Chen et al. (1993), J. Chromatog. A 652: 355-360 and Kambara et al. (1992), Electrophoresis 13: 542-546), ligands or haptens such as biotin, and radioactive isotopes such as 3H, 355, 32P 1251 and'4C.

EXPRESSION OF THE L66 PROTEIN/POLYPETIDE:
The nuclear receptor L66 nucleic acid or polypeptide may be synthesized in host cells transformed with a recombinant expression construct comprising a nucleic acid encoding the nuclear receptor L66.
Such a recombinant expression construct can also be comprised of a vector that is a replicable DNA construct.
Amplification vectors do not require expression control domains. All that is needed is fihe ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. See, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Press, New York, 1989).
An expression vector comprises a polynucleotide operatively linked to a prokaryotic promoter. Alternatively, an expression vector is a polynucleotide operatively linked to an enhancer-promoter that is a eukaryotic promoter, and the expression vector further has a polyadenylation signal that is positioned 3' of the carboxy-terminal amino acid and within a transcriptional unit of the encoded polypeptide. A
promoter is a region of a DNA molecule typically within about 500 nucleotide pairs in front of (upstream of) the point at which transcription begins (i.e., a transcription start site). In general, a vector contains a replicon and control sequences which are derived from species compatible with the host cell. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
Another type of discrete trahscription regulatory sequence element is an enhancer.
An enhancer provides specificity of time, location and expression level for a particular encoding region (e.g., gene). A major function of an enhancer is to increase the level of transcription of a coding sequence in a cell.

As used herein, the phrase "enhancer-promoter" means a composite unit that contains both enhancer and promoter elements. An enhancer-promoter is operatively linked to a coding sequence that encodes at least one gene product.
An enhancer-promoter used in a vector construct of the present invention may be any enhancer-promoter that drives expression in a prokaryotic or eukaryotic cell to be transformed/transfected.
A coding sequence of an expression vector is operatively linked to a transcription terminating region. RNA polymerase transcribes an encoding DNA sequence through a site where polyadenylation occurs.
An expression vector comprises a polynucleotide that encodes a nuclear receptor L66 polypeptide. Such a polynucleotide is meant to include a sequence of nucleotide bases encoding a nuclear receptor L66 polypeptide sufficient in length to distinguish said segment from a polynucleotide segment encoding a non- nuclear receptor polypeptide.
A polypeptide of the invention may also encode biologically functional polypeptides or peptides which have variant amino acid sequences, such as with changes selected based on considerations such as the relative hydropathic score of fihe amino acids being exchanged.
These variant sequences are those isolated from natural sources or induced in the sequences disclosed herein using a mutagenic procedure such as site-directed mutagenesis.
Furthermore, an expression vector of the present invention may contain regulatory elements for optimized translation of the polypeptide in prokaryotic or eukaryotic systems. This sequences are operatively located around the transcription start site and are most likely similar to ribosome recognition sites like prokaryotic ribosome binding sites (RBS) or eukaryotic Kozak sequences as known in the art (Kozak M., Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187-208 (1999).

An expression vector of the present invention is useful both as a means for preparing quantities of the nuclear receptor L66 polypeptide-encoding DNA itself, and as a means for preparing the encoded nuclear receptor L66 polypeptide and peptides.
It is contemplated that where nuclear receptor L66 polypeptides of the invention are made by recombinant means, one may employ either prokaryotic or eukaryotic expression vectors as shuttle systems.
Where expression of recombinant nuclear receptor L66 polypeptides is desired and a eukaryotic host is contemplated, it is most desirable to employ a vector such as a plasmid, that incorporates a eukaryotic origin of replication. Additionally, for the purposes of expression in eukaryotic systems, one desires to position the nuclear receptor L66 encoding sequence or if desired parts thereof (SEQ ID NO. 1, 4, 6, 8, 10, 12, 17, and/or 22) adjacent to and under the control of an effective eukaryotic promoter, To bring a coding sequence under control of a promoter, whether it is eukaryotic or prokaryotic, what is generally needed is to position the 5' end of the translation initiation site of the proper translational reading frame of the polypeptide between about 1 and about 2000 nucleotides 3' of or downsfiream with respect to the promoter chosen.
Furthermore, where eukaryotic expression is anticipated, one would typically desire to incorporate into the transcriptional unit which includes the nuclear receptor L66 polypeptide, an appropriate polyadenylation side.
The invention provides homogeneous compositions of mammalian nuclear receptor L66 polypeptide produced by transformed prokaryotic or eukaryotic cells as provided herein. Such homogeneous compositions are intended to be comprised of mammalian nuclear receptor L66 protein that comprises at least 90% of the protein in such homogenous composition. The invention also provides membrane preparation from cells expressing mammalian nuclear receptor L66 polypeptide as the result of transformation with a recombinant expression construct, as described here.
Within the scope of the present invention the terms recombinant protein or coding sequence both also include tagged versions of the protein depicted in SEQ ID
NO. 3, 19 and/or 22, and/or encoded by the nucleic acids according to the invention and fusion proteins of said proteins or parts thereof such as splice variants with any other recombinant protein. Tagged versions here means that small epitopes of 3-20 amino acids are added fio the original protein by extending the coding sequence either at the 5'or the 3'terminus leading to N-terminal or C-terminal extended proteins respectively, or that such small epitopes are included elsewhere in the protein. The same applies for fusion proteins where the added sequences are coding for longer proteins, varying between 2 and 100 kDa. Tags and fusion proteins are usually used to facilitate purification of recombinant proteins by specific antibodies or affinity matrices or to increase solubility of recombinanfi proteins within the expression host.
Fusion proteins are also of major use as essential parts of yeast two hybrid screens for interaction partners of recombinant proteins.
Tags used in the scope of the present invention may include but are not limited to the following: EEF (alpha Tubulin), B-tag (QYPALT), E tag (GAPVPYPDPLEPR) c-myc Tag (EQKLISEEDL), Flag epitope (DYKDDDDK, HA tag (YPYDVPDYA), 6 or 10 x His Tag, HSV (QPELAPEDPED), Pk-Tag (GKPIPNPLLGLDST), protein C
(EDQVDPRLIDGK), T7 (MASMTGGQQMG), VSV-G (YTDIEMNRLGK), Fusion proteines may include Thioredoxin, Glutathiontransferase (GST), Maltose binding Protein (MBP), Cellulose Binding protein (CBD), chitin binding protein, ubiquitin, the Fc part of Immunoglobulins, and the IgG binding domain of Staphylococcus aureus protein A. These examples of course are illustrative and not limiting and the standard amino acid one letter code was used above.
For expression of recombinant proteins in living cells or organisms, vector constructs harboring recombinant L66 nuclear receptor as set forth in SEQ ID NO. 1 or 17 are transformed or transfected into appropriate host cells. Preferably, a recombinant host cell of the present invention is transfected with a polynucleotide of SEQ ID
NO. 1, 4, 22 or 17.
Means of transforming or transfecting cells with exogenous polynucleotide such as DNA molecules are well known in the art and include techniques such as calcium-phosphate- or DEAE-dextran-mediated transfection, protoplast fusion, electroporation, liposome mediated transfection, direct microinjection and virus infection (Sambrook et al., 1989).

The most frequently applied technique for transformation of prokaryotic cells is transformation of bacterial cells after treatment with calciumchloride to increase permeability (Dagert & Ehrlich, 1979), but a variety of other methods is also available for one skilled in the art.
The most widely used method for transfection of eukaryotic cells is transfection mediated by either calcium phosphate or DEAE-dextran. Although the mechanism remains obscure, it is believed that the transfected DNA enters the cytoplasm of the cell by endocytosis and is transported to the nucleus. Depending on the cell type, up to 90% of a population of cultured cells may be transfected at any one time.
Because of its high efficiency, transfection mediated by calcium phosphate or DEAE-dextran is the method of choice for studies requiring transient expression of the foreign nucleic acid in large numbers of cells. Calcium phosphate-mediated transfection is also used to establish cell lines that integrate copies of the foreign DNA, which are usually arranged in head-to-tail tandem arrays into the host cell genome.
In the protoplast fusion method, protoplasts derived from bacteria carrying high numbers of copies of a plasmid of interest are mixed directly with cultured mammalian cells. After fusion of the cell membranes (usually with polyethylene glycol), the contents of the bacterium are delivered into the cytoplasm of the mammalian cells and the plasmid DNA is transported to the nucleus. Protoplast fusion is not as efficient as transfection for many of the cell lines that are commonly used for transient expression assays, but it is useful for cell lines in which endocytosis of DNA occurs inefficiently. Protoplast fusion frequently yields multiple copies of the plasmid DNA tandemly integrated into the host chromosome.
The application of brief, high-voltage electric pulses to a variety of mammalian and plant cells leads to the formation of nanometer sized pores in the plasma membrane.
DNA is taken directly into the cell cytoplasm either through these pores or as a consequence of the redistribution of membrane components that accompanies closure of the pores. Electroporation may be extremely efficient and may be used both for transient expression of cloned genes and for establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA.
Liposome transfection involves encapsulation of DNA and RNA within liposomes, followed by fusion of the liposomes with the cell membrane. The mechanism of how DNA is delivered into the cell is unclear but transfection efficiencies may be as high as 90%.
Direct microinjection of a DNA molecule into nuclei has the advantage of not exposing DNA to cellular compartments such as !ow-pH endosomes. Microinjection is therefore used primarily as a method to establish lines of cells that carry integrated copies of the DNA of interest.
The use of adenovirus as a vector for cell transfection is well known in the art.
Adenovirus vector-mediated cell transfection has been reported for various cells (Stratford-Perricaudet et al., 1992), A transfected cell may be prokaryotic or eukaryotic, transfection may be transient or stable. Where it is of interest to produce a full length human or mouse L66 protein, cultured mammalian mouse, or human cells are of particular interest.
!n another aspect, the recombinant host cells of the present invention are prokaryotic host cells. In addition to prokaryotes, eukaryotic microbes, such as yeast may also be used illustrative examples for suitable cells and organisms for expression of recombinant proteins are belonging to but not limited to the following examples:
Insect cells, such as Drosophila Sf21, SF9 cells or others, Expression strains of Escherichia coli, such as XL1 blue, BRL21, M15, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenlua polymorpha and Pichia pastoris strains, immortalized mammalian cell lines such as AtT-20, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COSM6, COS-7, 293 and MDCK
cells, BHK-21 cells, Att 20HeLa cells, HeK 294, T47 D cells and others.
Expression of recombinant proteins within the scope of this invenfiion can also be performed in vitro. This may occur by a two step procedure, thereby producing first mRNA by in vitro transcription of an apt polynucleotide construct followed by in vitro translation with convenient cellular extracts. These cellular extracts may be reticulocyte lysates but are not limited to this type. In vitro transcription may be performed by T7 or SP6 DNA polymerase or any other RNA polymerase which can recognize per se or with the help of accessory factors the promoter sequence contained in the recombinant DNA construct of choice. Alternatively one of the recently made available one step coupled transcription/translation systems may be used for in vitro translation of DNA coding for the proteins of this invention, e.g. from Roche Molecular Biochemicals. One illustrative but not limiting example for such a system is the TNT~ T7 Quick System by Promega.
Expression of recombinant proteins in transfected cell may occur constitutively or upon induction. Procedures depend on the Cell/vector combination used and are well known in the art. In all.cases, transfected cells are maintained for a period of time sufficient for expression of the recombinant L66 nuclear receptor protein. A
suitable maintenance time depends strongly on the cell type and organism used and is easily ascertainable by one skilled in the art. Typically, maintenance time is from about 2 hours to about 14 days. For the same reasons and for sake of protein stability and solubility incubation temperatures during maintenance time may vary from 20°C to 42 °C.
Recombinant proteins are recovered or collected either from the transfected cells or the medium in which those cells are cultured. Recovery comprises cell disruption, isolation and purification of the recombinant protein. Isolation and purification techniques for polypeptides are well-known in the art and include such procedures as precipitation, filtration, chromatography, elecfirophoresis and the like.
In a preferred embodiment, purification includes but is not limited to affinity purification of tagged or non-tagged recombinant proteins. This is a well established robust technique easily adapted to any tagged protein by one skilled in the art. For affinity purification of tagged proteins, small molecules such as gluthathione, maltose or chitin, specific proteins such as the IgG binding domain of Staphylococcus aureus protein A, antibodies or specific chelates which bind with high affinity to the tag of the recombinant protein are employed. For afFinity purification of non-tagged proteins 22_ .
specific monoclonal or polyclonal antibodies, which were raised against said protein, can be used. Alternatively immobilized specific interactors of said protein may be employed for affinity purification. Interactors include native or recombinant proteins as well as native or artificial specific low molecular weight ligands.
CHEMICAL SYNTHESIS OF THE POLYPEPTIDE ACCORDING TO THE
INVENTION:
Alternatively, the protein itself may be produced using chemical methods to synthesize any of the amino acid sequences according to the invention or that is encoded by the nucleotide sequences according to the invention (SEQ ID NO. 1, 4, 17 or 22) and/or a portion thereof and/or splice variants thereof. For example, peptide synthesis can be performed using conventional Merrifield solid phase f-Moc or t-Boc chemistry or various solid-phase techniques (Roberge, J. Y, et al. (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI
431A Peptide Synthesizer (Perkin Elmer). The newly synthesized peptides) may be substantially purified by preparative high perFormance liquid chromatography (e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure;
Creighton, supra). Additionally, the amino acid sequences according to the invention, i.e. SEQ ID NO. 3, 24 or 19 or the sequence that is encoded by SEQ ID NO. 1, 4, 17 or 22 or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
SCREENING ASSAYS
The invention also concerns a method for screening for agents which are capable of inhibiting the cellular function of the nuclear receptor L66 comprising the steps of contacting one or more candidate agents with a polypeptide according to the invention, removing unbound agents) and' detecting whether the agents) interact with the polypeptide of the nuclear receptor.

_ 23 The invention also concerns method for inhibiting the cellular function of the nuclear receptor L66, comprising the steps of contacting a cell with a binding agent of a polypeptide previously identified as outlined herein whereby the cellular function of L66 is inhibited.
Such a binding agent may be an antibody., RNA. an anti-sense oligonucleotide.
a ribozyme or one of substances shown below or identified in a respective assay as disclosed herein.
In still a further embodiment, the present invention concerns a method for identifying new nuclear receptor inhibitory or stimulatory substances, which may be termed as "candidate substances". It is contemplated that this screening technique proves useful in the general identification of compounds that serve the purpose of inhibiting or stimulating nuclear receptor activity.
In one embodiment of the invention the following subsfiances are disclosed as potential interactors of the nuclear receptor according to the invention:
Steroids: dexamethasone-t-butylacetate, RU486, progesterone, 17-aipha-hydroxyprogesterone, 1,16-alpha dimethylpregnenolone, 17-alpha-hydroxypregnenonlone, pregnenonlone, 5beta-pregnane-3,20-dione, pregnenonlone-16-carbonitrile, 5beta-pregnane-3,20-dione, androstanol, corticosterone, dehydroepiandrosterone, dihydroxytestosterone, estradiol, cortisol, cortisone, dihydroxytestosterone.
Other substances: transnonachlor, chlordane, spironolactone, cyproterone acetate, rifampicin, nefipine, diethylstilbestrol, coumesterol, clotrimazole, lovastatin, phenoarbital, pthalic acid, nonylphenol, 1,4-bis(2-(3,5-dichloropyridyloxy1))benzene, This also includes the use of heteromultimeric complexes of the nuclear receptor with other proteins, such as heterodimeric complexes with RXR, or any other binding partner.

Accordingly, in screening assays to identify pharmaceuticals agents which affect nuclear receptor activity, it is proposed that compounds isolated from natural sources, such as fungal extracts, plant extracts, bacterial extracts, higher eukaryotic cell extracts, or even extracts from animal sources, or marine, forest or soil samples, may be assayed for the presence of potentially useful pharmaceutical agents.
It will be understood that that the pharmaceutical agents to be screened could also be derived from chemical compositions or man-made compounds. The candidate substances can could also include monoclonal or polyclonal antibodies, peptides or proteins, such as those derived from recombinant DNA technology or by other means, including chemical peptide synthesis. The active compounds may include fragments or parts or derivatives of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive.
We anticipate that such screens will in some cases lead to the isolation of agonists of nuclear receptors, in other cases to the isolation of antagonists. In other instances, substances will be identified that have mixed agonistic and antagonistic effects, or affect nuclear receptors in any other way.
CELL BASED ASSAYS
To identify a candidate substance capable of influencing L66 nuclear receptor activity, one first obtains a recombinant cell line. One designs the cell line in such a way that the activity of the nuclear receptor leads to the expression of a protein which has an easily detectable phenotype ( a reporter), such as luciferase, fluorescent proteins such as green or red fluorescent protein, beta-galactosidase, alpha-galactosidase, beta-lactamase, chloramphenicol-acetyl-transferase, beta-glucuronidase, or any protein which can be detected by a secondary reagent such as an antibody.
Methods for detecting proteins using antibodies, such as ELISA assays, are well known to those skilled in the art.
Here, the amount of reporter protein present reflects the transcriptional activity of the nuclear receptor. This recombinant cell line is then screened for the effect of _25 substances on the expression of the reporters, thus measuring the effect of these substances on the activity of the nuclear receptor. These substances can be derived from natural sources, such as fungal extracts, plant extracts, bacterial extracts, higher eukaryotic cell extracts, or even extracts from animal sources, or marine, forest or soil samples, maybe assayed for the presence of potentially useful pharmaceutical agents. It will be understood that that the pharmaceutical agents to be screened may be derived from chemical compositions or man-made compounds.
The candidate substances can also include monoclonal or polyclonal antibodies, peptides or proteins, such as those derived from recombinant DNA technology or by other means, including chemical peptide synthesis. The active compounds may include fragments or parts or derivatives of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive.
In general the assay comprises, contacting a suitable cell containing a reporter under the control of the L66 nuclear receptor with a test compound, monitoring said host cell for the expression of the reporter gene, wherein expression of the reporter reflects the transcriptional activity of the nuclear receptor L66, and therefore reflects effects of the compound on the nuclear receptor.
In other embodiments of the invention assays are included where measuring the activity of a dimer of the nuclear receptor L66 and another protein, such as RXR
takes place. Further included are assays aiming at the identification of compounds which specifically influence only the monomeric, homodimeric or homomultimeric form of the nuclear receptor, or influencing only multimeric forms of the nuclear receptor. Such assays include measuring the effect of a compound on the nuclear receptor in the absence of a binding partner, and measuring the effect of a compound on the nuclear receptor in the presence of a binding partner, such as RXR. One skilled in the art will find numerous more assays which are equally covered by the invention.
A cell line where the activity of a nuclear receptor determines the expression of a reporter can be obtained by creating a fusion gene driving the expression of a fusion protein. consisting of the ligand-binding domain of the L66 nuclear receptor fused to the DNA binding domain of a transcription factor with a known specificity for a given DNA sequence (the binding site). This DNA sequence (the binding site) can then be inserted in one or multiple copies before (5') to the promoter driving the expression of the reporter. Transcription factors useful for this approach include bacterial proteins, such as IexA, yeast proteins, such as Gal4, mammalian proteins such as NFkappaB
or NFAT, the glucocorticoid receptor, the estrogen receptor, or plant proteins. The binding sites for these proteins can all be used in combination with the appropriate transcription factor to generate a useful reporter assay.
Another way to screen for inhibitors is to identify binding sites on DNA for the L66 nuclear receptor, and operatively link this binding site to a promoter operatively linked to a reporter gene. Included among others are binding sites for heterodimers of the L66 nuclear receptor with another protein, such as RXR.
Furthermore, transgenic animals described in the invention can be used to derive cell lines useful for cellular screening assays.
Cell lines useful for such an assay include many different kinds of cells, including prokaryotic, animal, fungal, plant and human cells. Yeasfi cells can be used in this assay, including Saccharomyces cerevisiae and Schizosaccharomyces pombe cells.
Another way to build cellular assays to measure the effect of compounds is the use of the yeast two hybrid system (see for example see, for example, U.S. Pat.
No.
5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol.
Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
(1993) Oncogene 8:1693-1696; PCT Publication No. WO 94/10300, and U.S. Pat.
No. 5,667,973), and or possible variants of the basic two hybrid system as discussed e.g in Vidal M, Legrain P, Nucleic Acids Res. 1999 Feb 15;27(4):919-29.
Briefly, the two hybrid assay relies on reconstituting in vivo a functional transcriptional activator protein from two separate fusion proteins. In particular, the method makes use of chimeric genes which express hybrid proteins. To illustrate, a first hybrid gene comprises the coding sequence for a DNA-binding domain of a transcriptional activator fused in frame to the coding sequence for a TI polypeptide. The second hybrid protein encodes a transcriptional activation domain fused in frame to a sample gene from a cDNA library. If the bait and sample hybrid proteins are able to interact, e.g., form a TI-dependent complex, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription of a reporter gene which is operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and expression of the reporter gene can be detected and used to score for the interaction of the TI and sample proteins.
In such assays, one primarily measures the effect of a compound on a given interaction involving the L66 nuclear receptor and a binding protein. In a preferred embodiment of the invention systems using other hosts such as prokaryotes as E.
coli, or eukaryotic mammalian cells are described.
Two hybrid systems using hybrid protein fusions with other proteins than transcription factors, including enzymes such as beta-galactosidase or dihydrofolate reductase may also be applied. These assays are useful both to monitor the effect of a compound, including peptides, proteins or nucleic acids on an interaction of a nuclear receptor with a given binding partner, as well as to identify novel proteins or nucleic acids interacting with the nuclear receptor.
Monitoring the influence of compounds on cells may be applied not only in basic drug screening, but also in clinical trials. In such clinical trials, the expression of a panel of genes may be used as a "read out" of a particular drug's therapeutic effect.
CELL-FREE ASSAYS
Recombinant forms of the polypeptide according to SEQ ID NO. 3, 24 or 19 or as encoded by the nucleic acids according to the invention can be used in cell-free screening assays aiming at the isolation of compounds affecting the activity of nuclear receptors. In such an assay, the nuclear receptor polypeptide is brought into contact with a substance to test if the substance has an effect on the activity of the L66 receptor.

_ 28 The detection of an interaction between an agent and a receptor may be accomplished through techniques well-known in the art. These techniques include but are not limited to centrifugation, chromatography, electrophoresis and spectroscopy. The use of isotopically labeled reagents in conjunction with these techniques or alone is also contemplated. Commonly used radioactive isotopes include 3H, 14C, . 2ZNa, 32P~ 33P~ 355 45Ca~ 60C0~ 1251, and 1311. Commonly used stable isotopes include '2H, .13 C, 15N, 1s0.
For example, if an agent binds to the receptor of the present invention, the binding may be detected by using radiolabeled agent or radiolabeled receptor. Briefly, if radiolabeled agent or radiolabeled receptor is utilized, the agent-receptor complex may be detected by liquid scintillation or by exposure to x-ray film or phosho-imaging devices.
One way to screen for substances affecting nuclear receptor activity is to measure the effect of the binding of nuclear receptors to ligands, such as cofactors, activators, repressors, DNA, RNA, proteins, antibodies, peptides or other substances, including chemical compounds known to affect receptor activity. Assays measuring the binding of a protein to a ligand are well known in the art, such as ELISA assays, FRET
assays, bandshift assays, plasmon-resonance based assays, scintilllation proximity assays, fluorescence polarization assays.
In one example, a mixture containing the L66 polypeptide, effector and candidate substance is allowed to incubate. The unbound effector is separable from any effector/receptor complex so formed. One then simply measures the amount of each (e.g., versus a control to which no candidate substance has been added). This measurement may be made at various time points where velocity data is desired.
From this, one determines the ability of the candidate substance to alter or modify the function of the receptor.
Numerous techniques are known for separating the effector from efFector/receptor complex, and all such methods are intended to fall within the scope of the invention.
This includes the use of thin layer chromatographic methods (TLC), HPLC, spectrophotometric, gas chromatographiclmass spectrophotometric or NMR

analyses. Another method of separation is to immobilize one of the binding partners on a solid support, and to wash away any unbound material. It is contemplated that any such technique may be employed so long as it is capable of differentiating between the effector and complex, and may be used to determine enzymatic function such as by identifying or quantifying the substrate and product.
A screening assay provides a L66 receptor under conditions suitable for the binding of an agent to the L66 receptor. These conditions include but are not limited to pH, temperature, tonicity, the presence of relevant cofactors, and relevant modifications to the polypeptide such as glycosylation or lipidation. It is contemplated that the receptor can be expressed 'and utilized in a prokaryotic or eukaryotic cell.
The host cell expressing the L66 receptor can be used whole or the receptor can be isolated from the host cell. The L66 receptor can be membrane bound in the membrane of the host cell or it can be free in the cytosol of the host cell. The host cell can also be fractionated into sub-cellular fractions where the receptor can be found. For example, cells expressing the receptor can be fractionated into the nuclei, the endoplasmic reticulum, vesicles, or the membrane surfaces of the cell.
pH is preferably from about a value of 6.0 to a value of about 8.0, more preferably from about a value of about 6.8 to a value of about 7:8, and most preferably, about 7.4. In a preferred embodiment, temperature is from about 20°C degrees to about 50°C degrees more preferably, from about 30°C degrees to about 40°C degrees and even more preferably about 37°C degrees. Osmolality is preferably from about 5 milliosmols per liter (mosm%L) to about 400 mosm/l, and more preferably, from about 200 milliosmols per liter to about 400 mosm/l and, even more preferably from about 290 mosm/L to about 310 mosm/L. The presence of cofactors can be required for the proper functioning of the L66 receptor. Typical cofactors include sodium, potassium, calcium, magnesium, and chloride. In addition, small, non-peptide molecules, known as prosthetic groups may also be required. Other biological conditions needed for receptor function are well-known in the art.
It is well-known in the art that proteins can be reconstituted in artificial membranes, vesicles or liposomes. (Danboldt et a1.,1990). The present invention contemplates that the receptor can be incorporated into artificial membranes, vesicles or liposomes. The reconstituted receptor can be utilized in screening assays.
It is further contemplated that a receptor of the present invention can be coupled to a solid support, e.g., to agarose beads, polyacrylamide beads, polyacrylic, sepharose beads or other solid matrices capable of being coupled to polypeptides. Well-known coupling agents include cyanogen bromide (CNBr), carbonyldiimidazole, tosyl chloride, diaminopimelimidate, and glutaraldehyde.
In a typical screening assay for identifying candidate substances, one employs the same recombinant expression host as the starting source for obtaining the receptor polypeptide, generally prepared in the form of a crude homogenate. Recombinant cells expressing the receptor are washed and homogenized to prepare a crude polypeptide homogenate in a desirable buffer such as disclosed herein. In a typical assay, an amount of polypeptide from the cell homogenate, is placed into a small volume of an appropriate assay buffer at an appropriate pH. Candidate substances, such as agonists and antagonists, are added to the admixture in convenient concentrations and the interaction between the candidate substance and the receptor polypeptide is monitored.
Where one uses an appropriate known substrate for the L66 receptor, one can, in the foregoing manner, obtain a baseline activity for the recombinantly produced receptor. Then, to test for inhibitors or modifiers of the receptor function, one can incorporate into the admixture a candidate substance whose effect on the L66 receptor is unknown. By comparing reactions which are carried out in the presence or absence of the candidate substance, one can then obtain information regarding the effect of the candidate substance on the normal function of the receptor.
Accordingly, this aspect of the present invention will provide those of skill in the art with methodology that allows for the identification of candidate substances having the ability to modify the action of nuclear receptor polypeptides in one or more manners.
Additionally, screening assays for the testing of candidate substances are designed to allow the determination of structure-activity relationships of agonists or antagonists with the receptors, e.g., comparisons of binding between naturally-occurring hormones or other substances capable of interacting with or otherwise modulating the receptor; or comparison of the activity caused by the binding of such molecules to the receptor.
In certain aspects, the polypeptides of the invention are crystallized in order to carry out x-ray crystallographic studies as a means of evaluating interactions with candidate substances or other molecules with the nuclear receptor polypeptide.
For instance, the purified recombinant polypeptides of the invention, when crystallized in a suitable form, are amenable to detection of intra-molecular interactions by x-ray crystallography. In another aspect, the structure of the polypeptides can be determined using nuclear magnetic resonance.
PHARMACEUTICAL COMPOSITION:
This invention provides a pharmaceutical composition comprising an effective amount of a agonist or antagonist drug identified by the method described herein and a pharmaceutically acceptable carrier. Such drugs and carrier can be administered by various routes, for example oral, subcutaneous, intramuscular, intravenous or intracerebral. The preferred route of administration would be oral at daily doses of about 0.01 -100 mg/kg.
This invention provides a method of treating metabolic disorders, immunological indications, hormonal dysfunctions, neurosystemic diseases wherein the abnormality is improved by reducing the activity of L66 receptor or blocking the binding of ligands to a L66 receptor, which method comprises administering an effective amount of the antagonist-containing pharmaceutical composition described above to suppress the subject's appetite. Similarly, the invention also provides methods for treating diseases and conditions resulting from metabolic disorders, immunological indications, hormonal dysfunctions, neurosystemic diseases, which method comprises administering an effective amount of an agonist-containing pharmaceutical composition described above.

_ 32 TRANSFORMATION OF CELLS AND DRUG SCREENING
The recombinant expression constructs of the present invention are useful in molecular biology to transform cells which do not ordinarily express L66 to thereafter express this receptor.
Such cells are useful as intermediates for making cellular preparations useful for receptor binding assays, which are in turn useful for drug screening. Drugs identified from such receptor assays can be used for the treatment of metabolic disorders, immunological indications, hormonal dysfunctions, and/or neurosystemic diseases.
The recombinant expression constructs of the present invention are also useful in gene therapy. Cloned genes of the present invention, or fragments thereof, may also be used in gene therapy carried out by homologous recombination or site-directed mutagenesis. See generally Thomas & Capecchi, Cell 51, 503-512 (1987);
Bertling, Bioscience Reports 7, 107-112 (1987); Smithies et al., Nature 317, 230-234 (1985).
Oligonucleotides of the present invention are useful as diagnostic tools for probing L66 expression in tissues. For example, tissues are probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiographic techniques, as explained in greater detail in the Examples below, to investigate native expression of this receptor or pathological conditions relating thereto. Further, chromosomes can be probed to investigate the presence or absence of the L66, and potential pathological conditions related thereto, as also illustrated by the Examples below. Probes according to the invention should generally be at least about 15 nucleotides in length to prevent binding to random sequences, but, under the appropriate circumstances may be smaller (see above for details on hybridization).

POLYPEPTIDE
Another aspect of the invention includes an antibody specifically reactive with the protein or any part of the protein according to the invention (SEQ ID NO. 3, 24 or 19) and or a polypeptide encoded by the nucleotide sequence of the nuclear receptor L66 (see also figures). (The term "antibody" refers to intact molecules as well as fragments thereof, such as Fa, F(ab)₂, and Fv, which are capable of binding the epitopic determinant.) By using immunogens derived from the polypeptide according to the invention (SEQ ID NO. 3, 24, 19) and/or encoded by the nucleic acids according to the invention, anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (E, Howell & D. Lane. Antibodies: A
Laboratory Manual. Cold Spring Harbor Laboratory (1988)).
A polyclonal antibody is prepared by immunizing a mammal, such as a mouse, a hamster or rabbit with an immunogenic form of the polypeptide, i.e. the human polypeptide of the present invention, and collecting antisera from that immunized animal. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
As an immunizing antigen, fusion proteins, intact polypeptides or fragments containing small peptides of interest can be used. They can be derived by expression from a cDNA transfected in a host cell with subsequent recovering of the protein/peptide or peptides can be synthesized chemically (e.g. oligopeptides with 10-15 residues in length). Important tools for monitoring the function of the gene according to the present invention, i.e. encoded by a sequence according to SEQ ID
NO. 1, 4, 24 or 17 (or portions thereof or splice variants thereof) are antibodies against various domains of the protein according to the invention. Various Oligopeptides from the N- and C-terminal sequences and the DBD/hinge region of the protein can be used as antigens.
A given polypeptide or polynucleotide may vary in its immunogenicity. It is often necessary to couple the immunogen (e.g. the polypeptide) with a carrier, Commonly used carriers that are chemically coupled to peptides include bovine serum albumin (BSA) and keyhole limpet hemocyanin (ICLH). The coupled peptide is then used to immunize the animal in the presence of an adjuvant, a non-specific stimulator of the immune response in order to enhance immunogenicity. The production of polyclonal antibodies is monitored by detection of antibody titers in plasma or serum at various time points following immunization. Standard ELISA or other immunoassays can be 34 _ used with the immunogen as antigen to assess the levels of antibodies. When a desired level of immunogenicity is obtained, the immunized animal may be bled and the serum isolated, stored and purified.
To produce monoclonal antibodies, antibody-producing cells (e.g. spleen cells) from an immunized animal (preferably mouse or rat) are fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Where the immunized animal is a mouse, a preferred myeloma cell is the murine NS-1 myeloma cell. Such techniques are well known in the art, and include, for example, the hybridoma technique (originally developed by Kohler &
Milstein.
Nature 256: 495-497 (1975)), the human B cell hybridoma technique (Kozbar et al.
Immunology Today 4:72 (1983)), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al. Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, Inc. pp. 77-96 (1985)).
The fused spleen/myeloma cells are cultured in a selective medium to select fused spleen/myeloma cells from the parental cells. Fused cells are separated from the mixture of non-fused parental cells, for example, by the addition of agents that block the de novo synthesis of nucleotides in the tissue culture media. This culturing provides a population of hybridomas from which specific hybridomas are selected.
Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants for reactivity with an antigen-polypeptide. The selected clones may then be propagated indefinitely to provide the monoclonal antibody in convenient quantity.
The creation of antibodies which specifically bind the polypeptide according to the invention (SEQ ID NO. 3, 24 or 19) and/or encoded by the nucleotide sequence of the nuclear receptor L66 or its complement (SEQ ID NO. 1, 4, 17 or 22) provides an important utility in immunolocalization studies, and may play an important role in the diagnosis and treatment of receptor disorders. The antibodies may be employed to identify tissues, organs, and cells which express or the nuclear receptor L66.
Antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate nuclear receptor L66 protein levels in tissue or from cells in bodily fluid as part of a clinical testing procedure.

Monoclonal antibodies provided by the present invention are also produced by recombinant genetic methods well known to those of skill in the art, and the present invention encompasses antibodies made by such methods that are immunologically reactive with an epitope of a mammalian nuclear L66 receptor protein or peptide.
The present invention encompasses fragments of the antibody that are immunologically reactive with an epitope of a mammalian nuclear L66 receptor protein or peptide. Such fragments are produced by any number of methods, including but not limited to proteolytic cleavage, chemical synthesis or preparation of such fragments by means of genetic engineering technology. The present invention also encompasses single-chain antibodies that are immunologically reactive with an epitope of a mammalian nuclear L66 receptor protein or peptide made by methods known to those of skilled in the art.
CHIMERIC ANTIBODIES AND OTHER TYPES OF ANTIBODIES:
The invention also includes chimeric antibodies, comprised of light chain and heavy chain peptides immunologically reactive to an epitope that is a mammalian nuclear L66 receptor protein or peptide. The chimeric antibodies embodied in the present invention include those that are derived from naturally occurring antibodies as well as chimeric antibodies made by means of genetic engineering technology well known to those of skill in the art.
Also included are methods for the generation of antibodies against L66 which rely on the use of phage display systems and related systems, such as described in Hoogenboom HR, de Bruine AP, Hufton SE, Hoet RM, Arends JW, Roovers RC, Immunotechnology 1998 Jun;4(1 ):1-20, and references therein.

The present invention also encompasses an epitope of a mammalian nuclear L66 receptor protein or peptide that is comprised of sequences and/or a conformation of sequences present in the mammalian nuclear L66 receptor protein or peptide molecule. This epitope may be naturally occurring, or may be the result of proteolytic cleavage of the mammalian nuclear L66 receptor protein or peptide molecule and isolation of an epitope-containing peptide or may be obtained by synthesis of an epitope-containing peptide using method of genetic engineering technology and synthesized by genetically engineered prokaryotic or eukaryotic cells.

Antisense oligonucleotides are short single stranded DNA or RNA molecules which may be used to block the availability of the L66 receptor messenger. Synthetic derivatives of ribonucleotides or deoxyribonucleotides and/or PNAs (see above) are equally possible.
The sequence of an antisense oligonucleotide is at least partially complementary to the sequence (or the gene) of interest. The complementarity of the sequence is in any case high enough to enable the antisense oligonucleotide to bind to the nucleic acid according to the invention or parts fihereof..Many examples exist in which the binding of oligonucleotides to the target sequence interFere with the biological function of the targeted sequence (Brysch W, Schlingensiepen KH, Design and application of antisense oligonucleotides in cell culture, in vivo, and as therapeutic agents, Cell Mol Neurobiol 1994 Oct;14(5):557-68; Wagner RW, Gene inhibition using antisense oligodeoxynucleotides, Nature 1994 Nov 24;372(6504):333-5 or Brysch W, Magal E, Louis JC, Kunst M, Klinger l, Schlingensiepen R, Schlingensiepen KH Inhibition of p185c-erbB-2 proto-oncogene expression by antisense oligodeoxynucleotides down-regulates p185-associated tyrosine-kinase activity and strongly inhibits mammary tumor-cell proliferation, Cancer Gene Ther 1994 Jun;1(2):99-105 or Monia BP, Johnston JF, Ecker DJ, Zounes MA, Lima WF, Freier SM Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides, J Biol Chem 1992 Oct 5;267(28):19954-62 or Bertram J, Palfner K, Killian M, Brysch W, Schlingensiepen KH, Hiddemann W, Kneba M, Reversal of multiple drug resistance in vitro by phosphorothioate oligonucleotides and ribozymes, Anticancer Drugs 1995 Feb;6(1):124-34) This interference occurs in most instances at the level of translation, i.e.
through the inhibition of the translational machinery by oligonucleotides fihat bind to mRNA, however, two other mechanisms of interference with a given gene's function by oligonucleotides can also be envisioned, (i) the functional interference with the transcription of a gene through formation of a triple helix at the level of genomic DNA
and the interference of oligonucleotides with the function of RNA molecules that are executing at least part of their biological function in the untranslated form (Kochetkova M, Shannon MF, Triplex-forming oligonucleotides and their use in the analysis of gene transcription. Methods Mol Biol 2000;130:189-201 Rainer B.
Lanz1, Neil J. McKenna1, Sergio A. Onate1, Urs Albrecht2, Jiemin Wong1, Sophia Y.
Tsai1, Ming-Jer Tsai1, and Bert W. O'Malley A Steroid Receptor Coactivator, SRA, Functions as an RNA and Is Present in an SRC-1 Complex Cell, Vol. 97, 17-27, April, 1999).
Antisense oligonucleotides can be conjugated to different other molecules in order to deliver them to the cell or tissue expressing L66. For instance the antisense oligonucleotide can be conjugated to a carrier protein (e.g. ferritin) in order to direct the oligonucleotide towards the desired target tissue, i.e. in case of ferritin predominantly to the liver.
Antisense expression constructs are expression vector systems that allow the expression - either inducible or uninducible - of a complementary sequence to the L66 sequences according to the invention. The potential possibility of such an approach has been demonstrated in many different model systems (von Ruden T, Gilboa E, Inhibition of human T-cell leukemia virus type I replication in primary human T cells that express antisense RNA, J Virol 1989 Feb;63(2):677-82; Nemir M, Bhattacharyya D, Li X, Singh K, Mukherjee AB, Mukherjee BB, Targeted inhibition of osteopontin expression in the mammary gland causes abnormal morphogenesis and lactation deficiency, J Biol Chem 2000 Jan 14;275(2):969-76; Ma L, Gauville C, Berthois Y, Millot G, Johnson GR, Calvo F,Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line, Oncogene 1999 Nov 11;18(47):6513-20; Refolo LM, Eckman C, Prada CM, Yager D, Sambamurti K, Mehta N, Hardy J, Younkin SG, Antisense-induced reduction of presenilin 1 expression selectively increases the production of amyloid beta42 in _ _ _ ~ 38 transfected cells, J Neurochem 1999 Dec;73(6):2383-8; Buckley NJ, Abogadie FC, Brown DA, Dayrell M, Caulfield MP, Delmas P, Haley JE, Use of antisense expression plasmids to attenuate G-protein expression in primary neurons, Methods Enzymol 2000;314:136-48).
According to the invention an antisense expression construct can be constructed with virtually any expression vector capable of fulfilling at least the basic requirements known to those skilled in the art.
In one embodiment of the invention retroviral expression systems or tissue specific gene expression systems are preferred.
Current standard technologies for delivering antisense constructs are perFormed through a conjugation of constructs with liposomes and related, complex-forming compounds, which are transferred via electroporation techniques or via particle-mediated "gene gun" technologies into the cell. Other techniques may be envisioned by one skilled in the art.
Microinjection still plays a major role in most gene transfer techniques for the generation of germ-line mutants expressing foreign DNA (including antisense RNA
constructs) and is preferred embodiment of the present invention.

Ribozymes are either RNA molecules (Gibson SA, Pellenz C, Hutchison RE, Davey FR, Shillitoe EJ, Induction of apoptosis in oral cancer cells by an anti-bcl-2 ribozyme delivered by an adenovirus vector, Clin Cancer Res 2000 Jan;6(1 ):213-22;
Folini M, Colella G, Villa R, Lualdi S, Daidone MG, Zaffaroni N, Inhibition of Telomerase Activity by a Hammerhead Ribozyme Targeting the RNA Component of Telomerase in Human Melanoma Cells, J Invest Dermatol 2000 Feb;114(2):259-267; Halatsch ME, Schmidt U, Botefur IC, Holland JF, Ohnuma T, Marked inhibition of glioblastoma target cell tumorigenicity in vitro by retrovirus-mediated transfer of a hairpin ribozyme against deletion-mutant epidermal growth factor receptor messenger RNA, J
Neurosurg 2000 Feb;92(2):297-305; Ohmichi T, Kool ET, The virtues of self binding:

high sequence specificity for RNA cleavage by self-processed hammerhead ribozymes, Nucleic Acids Res 2000 Feb 1;28(3):776-783) or DNA molecules (Li J, Zheng W, Kwon AH, Lu Y, In vitro selection and characterization of a highly efficient Zn(II)-dependent RNA-cleaving deoxyribozyme; Nucleic Acids Res 2000 Jan 15;28(2):481-488) that have catalytic activity. The catalytic activity located in one part of the RNA (or DNA) molecule can be "targeted" to a specific sequence of interest by fusing the enzymatically active RNA molecule sequence with a short stretch of RNA
(or DNA) sequence that is complementary to the L66 transcript. Such a construcfi will, when introduced into a cell either physically or via gene transfer of a ribozyme expression construct find the L66 sequence (our sequence of interest) and bind via its sequence-specific part to said sequence. The catalytic activity attached to the consfiruct, usually associated with a special nucleic acid structure (people distinguish so called "hammerhead" structures and "hairpin" structures), will then cleave the targeted RNA. The targeted mRNA will be destroyed and cannot be translated efficiently, thus the protein encoded by the mRNA derived from L66 will not be expressed or at least will be expressed at significantly reduced amounts.
In a preferred embodiment the invention covers inducible ribozyme constructs (Koizumi M, Soukup GA, Kerr JN, Breaker RR, Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP, Nat Struct Biol 1999 Nov;6(11 ):1062-1071 ).
In a further preferred embodiment the invention concerns the use of "bivalent"
ribozymes (multimers of catalytically active nucleic acids) as described in (Hamada M, Kuwabara T, Warashina M, Nakayama A, Taira K, Specificity of novel allosterically trans- and cis-activated connected maxizymes that are designed to suppress BCR-ABL expression FEBS Lett 1999 Nov 12;461(1-2);77-85).

Also provided by the present invention are non-human transgenic animals grown from germ cells transformed with the L66 nucleic acid sequence according to the invention and fihat express the L66 receptor according to the invention and offspring and descendants thereof. Also provided are transgenic non-human mammals comprising a homologous recombination knockout of the native L66 receptor, as well as transgenic non-human mammals grown from germ cells transformed with nucleic acid antisense to the L66 nucleic acid of the invention and offspring and descendants thereof. Further included as part of the present invention are transgenic animals which the native L66 receptor has been replaced with the human homolog. Of course, ofFspring and descendants of all of the foregoing transgenic animals are also encompassed by the invention.
Transgenic animals according to the invention can be made using well known 10 techniques with the nucleic acids disclosed herein. E.g., Leder et al., U.S. Patent Nos.4,736,866 and 5,175,383; Hogan et al., Manipulating the Mouse Embryo, A
Laboratory Manual (Cold Spring Harbor Laboratory (1986)); Capecchi, Science 244, 1288 (1989); Zimmer and Gruss, Nature 338, 150 (1989); Kuhn et al., Science 269, 1427 (1995); Katsuki et al., Science 241, 593 (1988); Hasty et al., Nature 350, 243 (1991); Stacey et al., Mot. Ceil Biol. 14, 1009 (1994); Hanks et al., Science 269, 679 (1995); and Marx, Science 269, 636 (1995). Such transgenic animals are useful for screening for and determining the physiological effects of L66 receptor agonists and antagonist.
20 Consequently, such transgenic animals are useful for developing drugs to regulate physiological activities in which L66 participates.
The following examples are provided for illustrative purposes only and are not intended, nor should they be construed, as limiting the invention in any manner.

The novel nuclear receptor sequences disclosed herein may be used for various in silico, i.e. compufier analyses. Such analyses may be for example nuclear receptor 30 specific sequence alignments which permit the identification of domains and even new receptors. The novel domain sequences disclosed herein may be used in order to create domain specific hidden markov models (hmms) or simply as search sequences.

_ . _ 41 In a preferred embodiment this similarity search tool is the BLAST algorithm.( Altschul, Stephen F., Warren Gish, Webb Miller, Eugene W. Myers, and David J.
Lipman (1990). Basic local alignment search tool. J. Mol. Biol. 215:403-10 and the sequence used is one of those disclosed herein.
Another search tool that may be used is FASTA (W. R. Pearson and D. J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444- 2448, and W. R. Pearson (1990) "Rapid and Sensitive Sequence Comparison with FASTP
and FASTA" Methods in Enzymology 183:63- 98).
The invention is not limited to one particular type of search tool. In one embodiment of the invention search tools are used that do not search by sequence similarity but by applying sequence profiles such as a profile generated when applying the Profile Hidden Markov Model.
Profile Hidden Markov Models also called "Hidden Markov Models", here abbreviated as HMM, are statistical models representing the consensus of the primary structure of a sequence family. The profiles use scores specific of the position of amino acids (or nucleotides) and position specific scores for the opening or the expansion of an insertion or deletion. Methods for the creation of profiles, starting from multiple alignments, have been introduced by Taylor (1986), Gribskov et al. (1987), Barton (1990) and Heinikoff (1996).
HMMs provide an utterly probabilistic description of profiles, i.e. Bayes' theory rules the positioning of all probability (evaluation) parameters (compare Krogh et al. 1994, Eddy 1996 and Eddy 1998). The central idea behind this is that a HMM is a finite model describing the probability distribution of an infinite number of possible sequences. The HMM consists of a number of states corresponding with the columns of a multiple alignment as it is usually depicted. Each state emits symbols (remainders) corresponding with the probability of the symbol emission (specific of the respective state), and the states are linked with each other by probabilities of the changing of states. Starting from one specific state, a succession of states is generated by changing from one state to the other in accordance with the probability '_ ' 42 of the changing of states, until a final state has been reached. Each state then emits symbols according to the probability distribution of emissions specific of this state, creating an observable sequence of symbols.
The attribute "hidden" has been derived from the fact that the underlying sequence of states cannot be observed. Only the sequence of symbols is visible. An assessment of the probabilities of changing of states and of emissions (the training of the model) is achieved by dynamic programming algorithms implemented in the HMMER
package.
The sequences according to the invention may be aligned with other nuclear receptor sequences in order to create a multiple sequence alignment which is used as the basis for the creation of a HMM.
If an existing HMM and a sequence are given, the probability that the HMM
could generate the sequence in question, can be calculated. The HMMER package provides a numerical quantity (the Score) in proportion to this probability, i.e. the information content of the sequence indicated as bits, measured according to the HMM.
See also Barton, G.J. (1990): Protein multiple alignment and flexible pattern matching, Methods Ezymol. 183: 403-427, Eddy, S.R. (1996): Hidden markov models. Curr. Opin. Strct. Biol. 6: 361-365, Eddy, S.R. (1998): Profile hidden markov models.Bioinformatics. 14: 755-763, Gribskov, M. McLachlan, A.D. and Eisenberg D.
(1987): Profile analysis: Detection of distantly related proteins. Proc. Natl.
Acad. Sci.
USA. 84: 4355-5358, Heinikoff, S. (1996): Scores for sequence searches and alignment, Curr. Opin. Strct. Biol. 6: 353-360, Krogh, A., Brown, M., Mian, I.S., Sjolander, K. and Haussler, D. (1994): Hidden markov models in computational biology: Applications to protein modelling. J. Mol. Biol. 235: 1501-1531, Taylor, W.R.
(1986): Identification of protein sequence homology by consensus template alignment. J. Mol. Biol. 188: 233-258.

In general the sequence are selected such that a query using a search sequence returns a result consisting of sequences which are at least to a certain degree similar to the query sequence.
In one embodiment of the invention amino acid sequences of the present invention are used to model the three-dimensional structure of the protein. Initially, this involves the comparison of the protein sequence with the sequence of related proteins where the structure is known, such as the human PPARy ligand-binding domain (Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV,Nature 1998 Sep 10;395(6698):137-43). The three-dimensionale structure can then be modelled using computer programs. From the three-dimensional structure, binding sites of potential inhibitors or activators can be predicted. It can further be predicted which kinds of molecule might bind there. The predicted substances can then be screened to test their effect on nuclear receptor activity.
EXAMPLES
EXAMPLE 1: CLONING AND EXPRESSION OF THE GENE ACCORDING TO THE
INVENTION
Construction of suitable vectors containing the desired coding and control sequences employs standard ligation and restriction techniques that are well undersfiood in the art. Isolated plasmids, DNA sequences, were synthesized oligonucleotides were cleaved, tailored, and religated in the form desired.
Site-specific DNA cleavage was performed by treatment with the suifiable restriction enzyme (or enzymes) under conditions that are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes.
See, e.g., New England Biolabs, Product Catalog. In general, about 1 pg of plasmid and/or DNA sequence was cleaved by one unit of enzyme in about 20 p1 of buffer solution. Often excess of restriction enzyme was used to ensure complete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37°C
are workable, although variations are tolerable.
After each incubation, protein was removed by extraction with phenol/chloroform, and may be followed by ether extraction. The nucleic acid was recovered from aqueous fractions by precipitation with ethanol. If desired, size separation of the cleaved fragments was performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzymology 65, 499-560 (1980).
Transformed host cells are cells which have been transformed or transfected with recombinant expression constructs made using recombinant DNA techniques and comprising mammalian nuclear receptor L66 encoding sequences. Preferred host cells for transient transfection are COS-7 cells. Transformed host cells may ordinarily express nuclear receptor L66, but host cells transformed for purposes of cloning or amplifying nucleic acid hybridization probe DNA need not express the nuclear receptor. When expressed, the mammalian nuclear L66 receptor protein was typically located in the host cell membrane.
Cultures of cells derived from multicellular organisms are desirable hosts for recombinant nuclear receptor L66 protein synthesis. In principal, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture.
However, mammalian cells are preferred. Propagation of such cells in cell culture has become a routine procedure. See Tissue Culture (Academic Press, Kruse &
Patterson, Eds., 1973). Examples of useful host cell fines are bacteria cells, insect cells, yeast cells, human 293 cells, VERO and HeLa cells, LMTK- cells, and W1138, BHK, COS-7, CV, and MDCK cell lines. Human 293 cells are preferred.
EXAMPLE 2: NUCLEAR RECEPTOR L66 TISSUE LOCALIZATION:
A multiple tissue northern blot (Clontech, Palo Alto) was hybridized to a labeled probe. The blot contained about 03 to 3 pg of poly A RNA derived from various tissues. Hybridization was carried out in a hybridization solution such as one __ - , 4g_ containing SSC (see Maniatis et al, ibid) at an optimized temperature between 50°c and 70°C, preferably 65°C. The filter was washed and a film exposed for signal detection (see also: Maniatis et al., Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.(1989)).
EXAMPLE 3: NUCLEAR RECEPTOR L66 cDNA ISOLATION FROM HUMAN AND
OTHER ORGANISMS:
A cloning strategy was used to clone the L66 receptor cDNA from specific cDNA
libraries (Clontech, Palo Alto) or alternatively, RNA was obtained from various tissues and used to prepare cDNA expression libraries by using for example an Invitrogen kit. (Invitrogen Corporation, San Diego). For the isolation of the L66 cDNA
clone the chosen library was screened under stringent condition (see definitions above) by using an L66 specific probe. The cDNA insert of positive clones was subsequently sequenced and cloned in a suitable expression vector.
Additionally, full length receptor L66 clones from human and mouse (Mus musculus) was obtained by using RACE PCR technology. In brief, suitable cDNA libraries were constructed or purchased. Following reverse transcription, the first strand cDNA was used directly in RACE PCR reactions using a RACE cDNA amplification kit according to the manufactures protocol (Clontech, Palo Alto). Amplified fragments were purified, cloned and subsequently used for sequence analysis.
To obtain information about the genomic organization (see also figures) of the receptor gene, genomic libraries were screened (commercially available clone libraries were used) with a receptor specific probe under stringent conditions.
Positive clones were isolated and the complete DNA sequence of the putative receptor region was determined by sequence analysis (Maniatis et al., Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.(1989)) (human genomic clone deposited at "Deutsche sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ, International Depositary Authority under the Budapest Treaty in Germany) under number DSM 14483).

EXAMPLE 4: NUCLEAR RECEPTOR L66 LIGAND BINDING ASSAY:
Saturation ligand binding analysis and ligand competition studies are carried out by using an over expressed L66 receptor-protein which is incubated with a labeled potential ligand at different concentrations. The L66 receptor may be immobilized on suitable surFaces. Various competitors are added in the presence of a labeled ligand.
Bound and unbound ligand is separated be using e.g. gel filtration or charcoal based methods or simply removed by a washing step if the receptor protein L66 is immobilized. Receptor-bound ligand is detected by scintilation counting.
EXAMPLE 5: NUCLEAR RECEPTOR L66 EXPRESSION ASSAY:
Relative quantification was performed in multiplex PCR reactions using the ABI
PRISM~ 7700 Sequence Detection System. Total RNA was used as template and was either ordered from Clontech or Ambion in the case of human normal tissue or was isolated from cell lines that were ordered from the DSMZ (German Collection of Microorganisms and Cell Cultures).
Relative quantification was achieved by normalising the results for the presence of 18S rRNA in the samples and subsequently by standardising the corresponding expression data to the expression levels detected in testis (testis = 1 ). A
standard curve was generated by diluting total RNA from testis tissue 1:10 fold, starting from 100 ng input amount and perForming 7 dilution steps. For the detection of target mRNA in other samples the input amount was always 100 ng total RNA, samples were measured in triplicates.
For the detection of 18S rRNA the endogenous control pre-developed assay reagent "Ribosomal RNA control (18S rRNA)" from Applied biosystems was used. For the detection of the target sequence, PCR primers and the probe were designed according to Applied biosystem's specifications using its Primer Express° software.
PCR primers were ordered from Interactiva (forward primer (SEQ ID NO. 14): CGT
GGG CTA ATG AAT TTT ACC AAG; reverse primer (SEQ ID NO. 15): GGC CCC

ATG GAG AAA TAT CAC T). The probe (SEQ ID NO. 16) CCA ATG AGG ATC AAA
CTG CAC TAC AGA AGG G was ordered from Applied Biosystems and was labelled with FAM at the 5'end and contained a quencher (TAMRA) at the 3' end.
EXAMPLE 6: FORMATION OF L66-RXR OR OTHER PROTEIN COMPLEXES
In order to explore the functional properties of L66, the DNA binding properties of L66 are analyzed. It has previously been shown that RXR is a common heterodimeric partner of various members of the nuclear receptor superfamily required for high affinity DNA binding (Hallenbeck et al., PNAS 89: 5572-5576 (1989)), Kliewer et al., EMBO J. 11: 1419-1435 (1992)). It has also been shown that DNA and ligand binding activities of the Dros. melanogaster ecdysan receptor (EcR) require heterodimer formation with RXR or USP (the homologue of RXR) (O'Malley in Endocrinology 125:
1119-1120 (1989)).
Consequently, it is of interest whether L66 can interact with RXR, or with other members of the family.
A two hybrid system is used. CV-1 cells are transiently transfected with cytomegalovirus promoter driven expression vectors containing the yeast GAL4 DNA
binding domain (DBD) alone, GAL4 linked to L66 LBD (LBD; i.e. GAL4-L66) and the 78 amino acid Herpes virus VP16 transactivation domain (VP) linked to the amon terminal end of the LBDs for human (or mouse) RXRa (VP-RXR), mouse PPARgamma (VP-PPAR) VDR (VP-VDR) and others.
All cells are cotransfected with a luciferase reporter construct containing about 4 copies of the yeast GAL4 upstream activating sequence and a ~i-galactosidase expression vector as internal control.
CV-1 cells are grown in DMEM supplemented with 10% AG1-X8 resin-charcoal stripped calf bovine serum, 50 U/ml penicillin G and 50 pg/ml streptomycin sulfate (DMEM-CBS) at 37°C in 5% C02. One day prior to transfection, cells are plated to 50-80% confluence using a phenol-red free DMEM with 10% resin charcoal stripped _ _ _ . 48 fetal bovine serum (DMEM-FBS). Cells are transfected (with reporter construct (300 ng/105 cells), cytomegalovirus driven receptor (100 ng/105 cells) and (i-galactosidase expression vectors (500 ng/10~ cells) by lipofection using N-~2-(2,3)-dioleoyloxy)propyl-N,N,N-trimethyl ammonium methyl sulfate} according to. the manufacturer s instructions (DOTAP, Boehringer Mannheim).
After 2 hours the liposomes are removed and cells treated for 40 hours with phenol-red free DMEM-FBS containing farnesol as the ligand. Cells are harvested and assayed for luciferase and (i-galactosidase activity.
All points are preferentially perFormed in triplicate and should ideally vary less than 10%. Experiments are repeated a 2-4 times. Data points are normalized for differences in transfection efficiency using ~i-galactosidase, and plotted as relative activity where the untreated reporter is defined to have an activity of 1 unit.
Neither the GAL4 DBD, nor the GAL4-L66 chimera should be capable of stimulating.
transcription from a reporter construct containing the GAL4 upstream activating sequence. Similarly, a fusion protein containing the Herpes virus VP16 firansactivation domain linked to the RXRa-LBD (VP-RXR) should be inactive when expressed alone or with the GAL4 DBD. However, when GAL4-L66 and VP-RXR are coexpressed, the reporter could be activated, indicating that L66 and RXRa interact efficiently in cells. Using similar VP16-LBD fusion proteins, interaction can be tested between L66 and receptors for peroxisome proliferators/fatty acids (PPAR), vitamin D3 (VDR), thyroid hormone, (T3R), retinoic acid (RAR), or other members of the nuclear receptor superfamily (see also US Pat. 6,005,086).
FIGURE CAPTIONS:
FIG. 1:
Fig. 1 shows the cDNA sequence of the L66 gene according to the invention. It also shows the reverse complement thereof.

___ _ _ __ _ -- 49 FIG. 2:
Fig. 1 shows the protein sequence of the L66 gene according to the invention.
FIG. 3:
Fig. 3 shows the domain composition of FXR-~i (L66) comprising a so called DNA
binding domain "DBD" and a ligand binding domain "LBD" which are present in members of the nuclear receptor superfamily. Also shown are the respective probability values for the presence of these domains which as can be seen in the figure are extremely high.
' FIG. 4:
Fig, 4 shows the cDNA sequence from L66 from Mus musculus (SEQ ID NO. 17) as well as its reverse complement (SEQ ID NO. 18) and the protein sequence of L66 from Mus musculus (SEQ ID NO. 19).
FIG. 5:
Fig. 5 shows the DNA sequence (NC Fragment) of a splice variant of L66 (SEQ ID
NO. 4). It also shows the reverse complement of the sequence (SEQ ID NO. 5).
FIG. 6: ' Fig. 6 shows the DNA sequence (PolyA1 Fragment, C-terminal end) of a splice variant of L66. It also shows the reverse complement of the sequence. This differs from the NC Fragment in so far as the poly-A-tail follows the exons 7 and 7B
directly (SEQ ID NO. 7).
FIG. 7:
Fig. 7 shows the DNA sequence (PolyA2 Fragment, C-terminal end) of a splice variant of L66. It also shows the reverse complement of the sequence. This differs __ _ , 50 ___ from the NC Fragment in so far as the poly-A-tail follows the exons 7 and 7B
directly (SEQ ID NO. 9).
FIG. 8:
Fig. 8 shows the DNA sequence (GF Fragment, C-terminal end) of a splice varianfi of L66. It also shows the reverse complement of the sequence. This differs from the NC
Fragment in so far as exon.8 is not present (SEQ ID NO. 11 ).
FIG. 9:
Fig. 9 shows the DNA sequence (SF Fragment, C-terminal end) of a splice variant of L66. It also shows the reverse complement of the sequence. This differs from the NC
Fragment in so far as exon 7 and 8 are not present (SEQ ID NO. 13).
FIG. 10:
Fig. 10 is a schematic representation of the exon distribution within the splice variants found.
FIG. 11:
Fig. 11 shows the result of a expression test on various cell lines and tissues.
FIG. 12:
Fig. 12 shows the genomic DNA sequence for L66 from Mus musculus (SEQ ID NO.
20).

__ " _ _ g1 _ FIG. 13:
Fig. 13 shows the reverse complement of the genomic DNA sequence for L66 from Mus musculus (SEQ ID NO. 21 ).
FIG. 14:
Fig. 14 shows a splice variant cDNA sequence of L66 from Mus musculus (SEQ ID
NO. 22), its reverse complement (SEQ ID NO. 23) and the corresponding protein sequence (SEQ ID NO. 24).
FIG. 15:
Fig. 15 lists all the sequences according to the invention, their respective origin ~as well as in which figures they are depicted.
FIG. 16:
Fig. 16 shows a BLASTP (improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements; Nucleic Acids Res. 2001 Jul 15;29(14):2994-3005.) - alignment of L66 against tremblnew (sequence identifier ~AF384555~AF384555) "NR1 H4"; product: "farnesol receptor"
from Homo sapiens farnesol receptor (NR1 H4) mRNA, complete cds, alternatively spliced. //:gp~AF384555~14326451 gene: "NR1H4"; product: "farnesol receptor";
Homo sapiens farnesol receptor (NR1 H4) mRNA, complete cds, alternatively spliced.
The hit is scoring at : 7e-88 (expectation value) for an alignment length (overlap) 389 having 46 % identities (Scoring matrix : BLOSUM62 (used to infer consensus pattern).
FiG. 17:

__ __52 Fig. 17 A shows the intron - exon composition of the mouse L66 gene (numericals).
Fig. 17 B shows the intron = exon composition of spliceform variant 1 of the mouse L,66 gene (numericals).
FIG. 18:
Fig. 18 A shows the intron - exon composition of the mouse L66 gene (sequences).
Fig. 18 B shows the intron - exon composition of spliceform variant 1 of the mouse L66 gene (sequences).
FIG. 19:
Fig. 19 shows a multiple sequence alignment of the L66 C4 zinc finger domain and shows the conservation of this domain.
FIG. 20:
Fig. 20 19 shows a multiple sequence alignment of the L66 protein and demonstrates the conservation of the L66 Ligand Binding Domain (LBD).

L-0020-01-WO-Ol.ST25 SEQUENCE LISTING
<110> LION Bioscience AG
<120> NOVEL MAMMALIAN NUCLEAR RECEPTOR L66 AND METHODS OF USE
<130> L-0020-01-WO-01 <160> 24 <170> PatentIn version 3.0 <210> 1 <211> 1152 <212> JNA
. . <:213=> . Horric.. sa~~i-ens -_ . _ ; . _ _ - . -<220>
<221> n <222> (1)..(1152) <223> n may be a, c, g or t <400> 1 cctggaataaaaaggtccagaccaacctattcttcctcgagaaataagggacaggaagaa60 ttctgtgtagtttgtggtgataaagcatcaccatcaccatatcattataatgcacttacc120 tgtgaaggttgcaaagaaatacctatggtaaaaaattttaaaacttttttattgggtttt180 tttcaatgtagcatcnnncaaaatgcagtatatagttgcaggaatggtagtcactgtgaa240 atggacatgtacatgcgtagaaaatgtcaagagtgcagactgaaaaagtataaggcagta300 ggaatgttggcagaatgtttgctcacagaaatccaatgtaaattaaagagacttcaaaag360 aactttaaggagaagaatcatttttactctaacatcaaagtggaagaggaaggagtagac420 cacagttttctatcatccaccactagacctggaaaagaaagcatggaactaactgaagag480 gaacatcagctcattaataacattgtggctgctcatcaaaaatataccattcctttagaa540 gaaacaaatttgtatctgcaggaacatacaaatcctgaactgagctttttgcaactctca600 gagacagcagtcctacacatacgtgggctaatgaattttaccaaggggctcccaggattt660 gaaaatttggccaatgaggatcaaactgcactacagaagggatcaaaaactgaagtgata720 Sei to L-0020-01-WO-01.ST25 tttctccatggggcccaactttacaatacaatgataatttccatatgtttgattctaccc780 tatgtttggatgaaaatacattttcgtatcagttttttgggtgttactgaagaatttatt840 acannnctgttttacttctacaaaagaatgagcaaacttgatgtaactaatactgaatat900 gctctgcttgcagcaacaattgttttttcagatcgtccatgccttaaaaataagcaatat960 atggaaaatttannngaaccagttttacaaatattgtataagtattcaaaaatgtatcat1020 ccagaagacccannncattttgcccatctcatatggaagcatactgaactgagaactctg1080 aattataaccattcagaaatacttagcacttggaaaacaaaggaccccaaattggctact1140 t~ta;:U:ctct~~ag . _. . , - . .. . . . . 1152.

<210> 2 <211> 1152 <212> DNA

<213> Homo sapiens <220>
<221> n <222> (1)..(1152) <223> n may be a, c> g or t <400> 2 ctcagagagtaaagtagccaatttggggtcctttgttttccaagtgctaagtatttctga60 atggttataattcagagttctcagttcagtatgcttccatatgagatgggcaaaatgnnn120 tgggtcttctggatgatacatttttgaatacttatacaatatttgtaaaactggttcnnn180 taaattttccatatattgcttatttttaaggcatggacgatctgaaaaaacaattgttgc240 tgcaagcagagcatattcagtattagttacatcaagtttgctcattcttttgtagaagta300 aaacagnnntgtaataaattcttcagtaacacccaaaaaactgatacgaaaatgtatttt360 catccaaacatagggtagaatcaaacatatggaaattatcattgtattgtaaagttgggc420 cccatggagaaatatcacttcagtttttgatcccttctgtagtgcagtttgatcctcatt480 ggccaaattttcaaatcctgggagccccttggtaaaattcattagcccacgtatgtgtag540 Seite 2 L-0020-01-WO-01.ST25 gactgctgtc tctgagagttgcaaaaagctcagttcaggatttgtatgttcctgcagata600 caaatttgtt tcttctaaaggaatggtatatttttgatgagcagccacaatgttattaat660 gagctgatgt tcctcttcagttagttccatgctttcttttccaggtctagtggtggatga720 tagaaaactg tggtctactccttcctcttccactttgatgttagagtaaaaatgattctt780 ctccttaaag ttcttttgaagtctctttaatttacattggatttctgtgagcaaacattc840 tgccaacatt cctactgccttatactttttcagtctgcactcttgacattttctacgcat900 gtacatgtcc atttcacagtgactaccattcctgcaactatatactgcattttgnnngat960 gctacatt~a aaaUaacccaataaaaaGgttttaaaattttttaccataggt~tttcttt.1020.

gcaaccttca caggtaagtgcattataatgatatggtgatggtgatgctttatcaccaca1080 aactacacag aattcttcctgtcccttatttctcgaggaagaataggttggtctggacct1140 ttttattcca gg 1152 <210>3 <211>384 <212>PRT

<213>Homo sapiens <220>.

<221>X

<222>(1)..(384) <223>X may be any amino acid <400>

ProGly IleLysArgSerArg ProThrTyr SerSerArg AsnLys Ser GlyGln GluGluPheCysVal ValCysGly LysAlaSer ProSer Asp ProTyr HisTyrAsnAlaLeu ThrCysGlu CysLysGlu IlePro Gly MetVal LysAsnPheLysThr PheLeuLeu PhePheGln CysSer Gly Sei to 3 L-0020-01-WO-01.ST25 Ile Xaa Gln Asn Ala Val Tyr Ser Cys Arg Asn Gly Ser His Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Tyr Lys Ala Ual Gly Met Leu Ala Glu Cys Leu Leu Thr Glu Ile Gln Cys Lys Leu Lys Arg Leu Gln Lys Asn Phe Lys Glu Lys Asn His Phe Tyr Ser Asn Ile Lys Val Glu Glu Glu Gly Val Asp His Ser Phe Leu .. ~ 13g . ...' ..: ' . 1. Z5. , :: .. . .. 140, ' ' . . ..
Ser Ser Thr Thr Arg Pro Gly Lys Glu Ser Met Glu Leu Thr Glu Glu Glu His Gln Leu Ile Asn Asn Ile Val Ala Ala His Gln Lys Ty r Thr Tle Pro Leu Glu Glu Thr Asn Leu Tyr Leu Gln Glu His Thr Asn Pro Glu Leu Ser Phe Leu Gln Leu Ser Glu Thr Ala Val Leu His Ile Arg Gly Leu Met Asn Phe Thr Lys Gly Leu Pro Gly Phe Glu Asn Leu Ala Asn Glu Asp Gln Thr Ala Leu Gln Lys Gly Ser Lys Thr Glu Val Ile Phe Leu His Gly Ala Gln Leu Tyr Asn Thr Met Ile Ile Ser Ile Cys Leu Ile Leu Pro Ty r Val Trp Met Lys Ile His Phe Arg Ile Ser Phe Leu Gly Val Thr Glu Glu Phe Iie Thr Xaa Leu Phe Tyr Phe Tyr Lys Arg Met Ser Lys Leu Asp Val Thr Asn Thr Glu Tyr Ala Leu Leu Ala Seite 4 L-0020-01-WO-01.ST25 Ala Thr Ile Ual Phe Ser Asp Arg Pro Cys Leu Lys Asn Lys Gln Tyr Met Glu Asn Leu Xaa Glu Pro Ual Leu Gln Ile Leu Tyr Lys Tyr Ser Lys Met Tyr His Pro Glu Asp Pro Xaa His Phe Ala His Leu Ile Trp Lys His Thr Glu Leu Arg Thr Leu Asn Tyr Asn His Ser Glu Ile Leu Ser Thr Trp Lys Thr Lys Asp Pro Lys Leu Aia Thr Leu Leu Ser Giu ..<L10> 4 - . . ... . ' , . . , ' : - ' - .- , <211> 2077 <212> DNA

<213> Homo sapiens <400> 4 cctggaataa aaaggtccag accaacctat tcttcctcga gaaataaggg acaggaagaa 60 ttctgtgtag tttgtggtga taaagcatca ccatcaccat atcattataa tgcacttacc 120 tgtgaaggtt gcaaagcatc aacaaaatgc agtatatagt tgcaggaatg gtagtcactg 180 tgaaatggac atgtacatgc gtagaaaatg tcaagagtgc agactgaaaa agtataaggc 240 agtaggaatg ttggcagaat gtttgcacac agaaatccaa tgtaaattaa agagacttca 300 aaagaacttt aaggagaaga atcattttta ctctaacatc aaagtggaag aggaaggagt 360 agaccacagt tttctatcat ccaccactag acctggaaaa gtgattcagg aaagcatgga 420 actaactgaa gaggaacatc agctcattaa taacattgtg gctgctcatc aaaaatatac 480 cattccttta gaagaaacaa atttctgcag gaacatacaa atcctgaact gagctttttg 540 caactctcag agacagcagt cctacacata cgtgggctaa tgaattttac caaggggctc 600 ccaggatttg aaaatttggc caatgaggat caaactgcac tacagaaggg atcaaaaact 660 gaagtgatat ttctccatgg ggcccaactt tacagtcaga aacaatcagc ctctgaaagt 720 tctgtgagaa tattaaatca ttcagattat acaccaaatt gtcacaatag gagtggtgat 780 Seite 5 L- 0020-01-WO-01.ST25 agaagtcttatttgttctatggaaaaattttacaatgaagaatgtccttctactactcta840 attggtttcaagaagctcatctggaaaatggtgataataatagaacttacctcatacagt900 attgtgactactacataaaataatacatagagatcgtccatgccttaaaaataagcaata960 tatggaaaatttacaagaaccagttttacaaatattgtataagtattcaaaaatgtatca1020 tccagaagacccatagcattttgcccatctcatatggaagcatactgaactgagaac 1077 <210> 5 <211> 1077 <212> DNA
<213> Homo Sapiens <400> 5 gttctcagtt cagtatgctt ccatatgaga tgggcaaaat gctatgggtc ttctggatga 60 tacatttttg aatacttata caatatttgt aaaactggtt cttgtaaatt ttccatatat 120 tgcttatttttaaggcatggacgatctctatgtattattttatgtagtagtcacaatact180 gtatgaggtaagttctattattatcaccattttccagatgagcttcttgaaaccaattag240 agtagtagaaggacattcttcattgtaaaatttttccatagaacaaataagacttctatc300 accactcctattgtgacaatttggtgtataatctgaatgatttaatattctcacagaact360 ttcagaggctgattgtttctgactgtaaagttgggccccatggagaaatatcacttcagt420 ttttgatcccttctgtagtgcagtttgatcctcattggccaaattttcaaatcctgggag480 ccccttggta aaattcatta gcccacgtat gtgtaggact gctgtctctg agagttgcaa 540 aaagctcagt tcaggatttg tatgttcctg cagaaatttg tttcttctaa aggaatggta 600 tatttttgat gagcagccac aatgttatta atgagctgat gttcctcttc agttagttcc 660 atgctttcct gaatcacttt tccaggtcta gtggtggatg atagaaaact gtggtctact 720 ccttcctctt ccactttgat gttagagtaa aaatgattct tctccttaaa gttcttttga 780 agtctcttta atttacattg gatttctgtg tgcaaacatt ctgccaacat tcctactgcc 840 ttatactttt tcagtctgca ctcttgacat tttctacgca tgtacatgtc catttcacag 900 Seite 6 L-0020-01-WO-01.ST25 tgactaccat tcctgcaact atatactgca ttttgttgat gctttgcaac cttcacaggt 960 aagtgcatta taatgatatg gtgatggtga tgctttatca ccacaaacta cacagaattc 1020 ttcctgtccc ttatttctcg aggaagaata ggttggtctg gaccttttta ttccagg 1077 <210>6 <211>331 <2l2>DNA

<213>Homo sapiens <220>
<221> n <222> (1)..(331) .. . <223> ~~n., may, be ,~. ~. u:.:or l~ .. ~ . . . . -<400>

cccaactttacagtcagaaacaatcagcctctgaaagttctgtgagaatattaaatcatt60 cagattatacaccaaattgtcacaataggagtggtgatagaagtcttatttgttctatgg120 aaaaattttacaatgaagaatgtccttctactactctaattggtaatatgactcaatatg180 aaatattatattggatgcaaaaatttgtatataatgtttaactttcttatactgctttga240 gatacaatgataatttccatatgtttgattctaccctatgtttggatgaaaatacatttt300 cgtatcngcccaaaaaaaaaaaaaaaaaaaa 331 <210>7 <211>331 <212>DNA

<213>Homo sapiens <220>
<221> n <222> (1)..(331) <223> "N" may be either A. C. G or T
<400> 7 tttttttttt tttttttttt gggcngatac gaaaatgtat tttcatccaa acatagggta 60 gaatcaaaca tatggaaatt atcattgtat ctcaaagcag tataagaaag ttaaacatta 120 Seite 7 L-0020-01-WO-01.ST25 tatacaaatt tttgcatcca atataatatt tcatattgag tcatattacc aattagagta 180 gtagaaggac attcttcatt gtaaaatttt tccatagaac aaataagact tctatcacca 240 ctcctattgt gacaatttgg tgtataatct gaatgattta atattctcac agaactttca 300 gaggctgatt gtttctgact gtaaagttgg g 331 <210>8 <211>311 <212>DNA

<213>Homo Sapiens <4fl0> ... . ...., .:. ~ . .. .
g ..~

cccaactttacagtcagaaacaatcagcctctgaaagttctgtgagaatattaaatcatt60 cagattatacaccaaa,ttgtcacaataggagtggtgatagaagtcttatttgttctatgg120 aaaaattti;acaatgaagaatgtccttctactactctaattggtaatatgactcaatatg180 aaatattatattggatgcaaaaatttgtatataatgtttaactttcttatactgctttga240 gatacaatgataatttccatatgtttgattctaccctatgtttggatgaggaaaaaaaaa300 aaaaaaaaaa a 311 <210>9 <211>311 <212>DNA

<213>Nomo sapiens <400>

ttttttttttttttttttttcctcatccaaacatagggtagaatcaaacatatggaaatt60 atcattgtatctcaaagcagtataagaaagttaaacattatatacaaatttttgcatcca120 atataatatttcatattgagtcatattaccaattagagtagtagaaggacattcttcatt180 gtaaaatttttccatagaacaaataagacttctatcaccactcctattgtgacaatttgg240 tgtataatctgaatgatttaatattctcacagaactttcagaggctgattgtttctgact300 gtaaagttggg 311 Seite 8 L-0020-01-WO-Ol.ST25 <210>10 <211>425 <212>DNA

<27.3>Homo sapi ens <400> IO
ggccaatgag gatcaaactg cactacagaa gggatcaaaa actgaagtga tatttctcca 60 tggggcccaa ctttacagtc agaaacaatc agcctctgaa agttctgtga gaatattaaa 120 tcattcagat tatacaccaa attgtcacaa taggagtggt gatagaagtc ttatttgttc 180 tatggaaaaa ttttacaatg aagaatgtcc ttctactact ctaattgatc gtccatgcct 240 taaaaataag-caatatatgg aaaatttaca agaaccagti ttacaaatat-tgtatuagta 30tJ.
ttcaaaaatg tatcatccag aagacccata gcattttgcc catctcatat ggaagcatac 360 tgaactgaga actctgaatt ataaccattc agaaatactt agcacttgga aaacaaagga 420 cccca 425 <210>11 <211>425 <212>DNA

<213>Homo Sapiens <400> 11 tggggtcctt tgttttccaa gtgctaagta tttctgaatg gttataattc agagttctca 60 gttcagtatg cttccatatg agatgggcaa aatgctatgg gtcttctgga tgatacattt 120 ttgaatactt atacaatatt tgtaaaactg gttcttgtaa attttccata tattgcttat 180 ttttaaggca tggacgatca attagagtag tagaaggaca ttcttcattg taaaattttt 240 ccatagaaca aataagactt ctatcaccac tcctattgtg acaatttggt gtataatctg 300 aatgatttaa tattctcaca gaactttcag aggctgattg tttctgactg taaagttggg 360 ccccatggag aaatatcact tcagtttttg atcccttctg tagtgcagtt tgatcctcat 420 tggcc 425 Seite 9 L-0020-01-WO-01.ST25 <210>12 <211>299 <212>DNA

<213>Homo Sapiens <400> 12 .
ggccaatgag gatcaaactg cactacagaa gggatcaaaa actgaagtga tatttctcca 60 tggggcccaa ctttacagtc agaaacaatc agcctctgaa aatcgtccat gccttaaaaa 120 taagcaatat atggaaaatt tacaagaacc agttttacaa atattgtata agtattcaaa 180 aatgtatcat ccagaagacc catagcattt tgcccatctc atatggaagc atactgaact 240 gagaactctg aattataacc attcagaaat acttagcact tggaaaacaa aggacccca 299 <210>13 <211>299 <212>DNA

<213>Nomo Sapiens <400> 13 tggggtcctt tgttttccaa gtgctaagta tttctgaatg gttataattc agagttctca 60 gttcagtatg cttccatatg agatgggcaa aatgctatgg gtcttctgga tgatacattt 120 ttgaatactt atacaatatt tgtaaaactg gttcttgtaa attttccata tattgcttat 180 ttttaaggca tggacgattt tcagaggctg attgtttctg actgtaaagt tgggccccat 240 ggagaaatat cacttcagtt tttgatccct tctgtagtgc agtttgatcc tcattggcc 299 <210> 14 <211> 24 <212> DNA
<213> Artificial <400> 14 cgtgggctaa tgaattttac caag 24 <210> 15 <211> 22 <212> DNA
<213> Artificial Seite 10 L-0020-Ol-WO-Ol.ST25 <400> 15 ggccccatgg agaaatatca ct 22 <210>16 <211>31 <212>DNA

<213>Artificial <400> 16 ccaatgagga tcaaactgca ctacagaagg g 31 <210> _ <~.~~> - .. . . _. . . . , . ~, ~:,n <212>
DNA

<213> musculus Mus <400>

atgttaataaaaccagatattttgccagaacaattccattatcagctgtgtgatacagat60 ttccaagaaccaccctattgtcaatattctaccgctcagtttcctccagcgttacagtcc120 ccatctttacaaagtcatttcaacacacatggcttggatccacagtacagtggaggcagt180 , tggtgtggactcgacgctcgagaatctggtcagtccacttatgtggttgttcacgatgat240 gaagatgaattccctggggcacaaaggtgcagagcaacttgttctttacgctggaagggt300 caagatgacatgctctgcatggtctgcggtgataaggcatcaggatatcactacaatgca360 cttacttgtgaggggtgcaaaggctttttccggcgtagcattaccaagaatgcagtgtat420 tcttgcaagaacggtggtcactgtgaaatggacatgtacatgcgcagaaaatgccaagag480 tgcagactgaagaagtgtaaggcggtggggatgttggcagaatgtttgctcacagagatc540 cagtgtaagtcaaagagacttcgcaagaacttcaagcacgggcctgccctgtaccctgcc600 atccaagtggaagatgaaggagcagacaccaaacacgtgtcatccagcaccagatctggg660 aaaggggttcaggacaacatgactctaactcaagaggaacatcggcttctgaataccata720 gtgactgctcaccaaaaatccatgattcccttgggagaaacaagcaaacttctgcaggag780 ggttccaaccccgaactaagttttctgagactctcagaggtatcagtcctgcacatacaa840 Seite L-0020-01-WO-01.ST25 gggctaatgaagtttaccaagggactcccaggatttgaaaatttaaccactgaggatcag900 gctgcattacagaaggcgtcaaaaactgaagtgatgttccttcatgtagcccagctttat960 ggtgggaaagactcaacctctggaagtactatgagaccagcaaagccctcagctgggaca1020 ctagaggtgcataatcctagcgctgatgaaagtgttcattctccggaaaactttctcaag1080 gaaggctacccttcggctcctctaactgatattactaaagaatttattgcctcactatct1140 tacttctaca gaagaatgag tgaacttcat gtatcggata ctgaatatgc tctgcttacg 1200 gcgacaacag tgcttttctc agatcgtcca tgccttaaaa ataagcagca tatagaaaac 1260 ._ ct~~aagaac c~c~tcctgca- acttttg~tt aagttttcaa .aaat gtacca tcca.g~agac ~-13?_~,,~
ccacagcatt tcgcccacct catagggagg cttactgaac tgagaactct gagtcacagc 1380 cactctgaaa tccttcgcat gtggaaaaca aaggacccca ggttggtgat gttattctct 1440 gagaaatggg atctgcactc attttcctga 1470 <210>18 <211>1470 <212>DNA

<213>Mus musculus <400> 18 tcaggaaaat gagtgcagat cccatttctc agagaataac atcaccaacc tggggtcctt 60 tgttttccac atgcgaagga tttcagagtg gctgtgactc agagttctca gttcagtaag 120 cctccctatg aggtgggcga aatgctgtgg gtcttctgga tggtacattt ttgaaaactt 180 aaacaaaagt tgcaggactg gttcttgtag gttttctata tgctgcttat ttttaaggca 240 tggacgatct gagaaaagca ctgttgtcgc cgtaagcaga gcatattcag tatccgatac 300' atgaagttca ctcattcttc tgtagaagta agatagtgag gcaataaatt ctttagtaat 360 atcagttaga ggagccgaag ggtagccttc cttgagaaag ttttccggag aatgaacact 420 ttcatcagcg ctaggattat gcacctctag tgtcccagct gagggctttg ctggtctcat 480 agtacttcca gaggttgagt ctttcccacc ataaagctgg gctacatgaa ggaacatcac 540 Seite 12 L-0020-01-WO-01.ST25 ttcagtttttgacgccttctgtaatgcagcctgatcctcagtggttaaattttcaaatcc600 tgggagtcccttggtaaacttcattagcccttgtatgtgcaggactgatacctctgagag660 tctcagaaaacttagttcggggttggaaccctcctgcagaagtttgcttgtttctcccaa720 gggaatcatggatttttggtgagcagtcactatggtattcagaagccgatgttcctcttg780 agttagagtcatgttgtcctgaacccctttcccagatctggtgctggatgacacgtgttt840 ggtgtctgctccttcatcttccacttggatggcagggtacagggcaggcccgtgcttgaa900 gttcttgcgaagtctctttgacttacactggatctctgtgagcaaacattctgccaacat960 ceccaccgcc.:ttu~acttct~tcagt-ctgca-~tcttggcattttctgcgcatg~Lacatgtc..1-t~20. -.. .

catttcacagtgaccaccgttcttgcaagaatacactgcattcttggtaatgctacgccg1080 gaaaaagcctttgcacccctcacaagtaagtgcattgtagtgatatcctgatgccttatc1140 accgcagaccatgcagagcatgtcatcttgacccttccagcgtaaagaacaagttgctct1200 gcacctttgtgccccagggaattcatcttcatcatcgtgaacaaccacataagtggactg1260 accagattctcgagcgtcgagtccacaccaactgcctccactgtactgtggatccaagcc1320 atgtgtgttgaaatgactttgtaaagatggggactgtaacgctggaggaaactgagcggt1380 agaatattgacaatagggtggttcttggaaatctgtatcacacagctgataatggaattg1440 ttctggcaaaatatctggttttattaacat 1470 <210>19 <211>489 <212>PRT

<213>Mus musculus <400> 19 Met Leu Ile Lys Pro Asp Iie Leu Pro Giu G1n Phe His Tyr Gin Leu Cys Asp Thr Asp Phe Gln Glu Pro Pro Tyr Cys Gln Tyr Ser Thr Ala Seite 13 L-0020-01-WO-01.ST25 Gln Phe Pro Pro Ala Leu Gln Ser Pro Ser Leu Gln Ser His Phe Asn Thr His Gly Leu Asp Pro Gln Tyr Ser Gly Gly Ser Trp Cys Gly Leu Asp Ala Arg Glu Ser Gly Gln Ser Thr Tyr Ual Val Ual His Asp Asp Glu Asp Glu Phe Pro Gly Ala Gln Arg Cys Arg Ala Thr Cys Ser Leu Arg Trp Lys Gly Gln Asp Asp Met Leu Cys Met Ual Cys Gly Asp Lys ._ .,qlU ser C;ly iyr:His-..~wr~;'~~n~Ala Leu Thr Cys Glu Gly. Cys.Lys G1~<:.-., ... . ._.

Phe Phe Arg Arg Ser Ile Thr Lys Asn Ala Ual Tyr Ser Cys Lys Asn Gly Gly His Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys.Lys Cys Lys Ala Ual Gly Met Leu Ala Glu Cys Leu Leu Thr Glu Ile Gln Cys Lys Ser Lys Arg Leu Arg Lys Asn Phe Lys His Gly Pro Ala Leu Tyr Pro Ala Ile Gln Val Glu Asp Glu Gly Ala Asp Thr Lys His Ual Ser Ser Ser Thr Arg Ser Gly Lys Gly Ual Gln Asp Asn Met Thr Leu Thr Gln Glu Glu His Arg Leu Leu Asn Thr Ile Ual Thr Ala His Gln Lys Ser Met Ile Pro Leu Gly Glu Thr Ser Lys Leu Leu Gln Glu Gly Ser Asn Pro Glu Leu Ser Phe Leu Arg Leu Ser . 260 265 270 Glu Ual Ser Ual Leu His Ile Gln Gly Leu Met Lys Phe Thr Lys Gly Seite 14 L-0020-01-WO-01.ST25 Leu Pro Gly Phe Glu Asn Leu Thr Thr Glu Asp Gln Ala Ala Leu Gln Lys Ala Ser Lys Thr Glu Ual Met Phe Leu His Ual Ala Gln Leu Tyr Gly Gly Lys Asp Ser Thr Ser Gly Ser Thr Met Arg Pro Ala Lys Pro Ser Ala Gly Thr Leu Glu Ual His Asn Pro Ser Ala Asp Glu Ser Ual His Ser Pro Glu Asn Phe Leu Lys Glu Gly Tyr Pro Ser Ala Pro Leu Thr Asp Ile Thr Lys Glu Phe Ile Ala Ser Leu Ser Tyr Phe Tyr Arg Arg Met Ser Glu Leu His Val Ser Asp Thr Glu Tyr Ala Leu Leu Thr Ala Thr Thr Ual Leu Phe Ser Asp Arg Pro Cys Leu Lys Asn Lys Gln His Ile Glu Asn Leu Gln Glu Pro Ual Leu Gln Leu Leu Phe Lys Phe Ser Lys Met Tyr His Pro Glu Asp Pro Gln His Phe Ala His Leu Ile Gly Arg Leu Thr Glu Leu Arg Thr Leu Ser His Ser His Ser Glu Ile Leu Arg Met Trp Lys Thr Lys Asp Pro Arg Leu Ual Met Leu Phe Ser Glu Lys Trp Asp Leu His Ser Phe Ser <210>20 <211>16200 <212>DNA

<213>Mus musculus <400> 20 ctttggggtc atagcattta ttaggtattg ttagttccat ttactgtgtt cctattgtgc 60 Seite 15 L-0020-01-WO-01.ST25 tggacggcaa gtcctgagatggaatagatgtgttgaactaactttctgttgagtataaaa120 cattttgagg tttaaaacaattatgaacttttttgaccttttatgatttgttctttttta180 tgttaataaa accagatattttgccagaacaattccattatcagctgtgtgatacagatt240 tccaagaacc accctattgtcaatattctaccgctcagtttcctccagcgttacagtccc300 catctttaca aagtcatttcaacacacatggcttggatccacagtacagtggaggcagtt360 ggtgtggact cgacgctcgagaatctggtcagtccacttatgtggttgttcacgatgatg420 aagatgaatt ccctggggcacaaaggtgcagagcaacttgttctttacgctggaagggtc480 ~- w aa~a~tgacat~gct~tg~ag ttgcgg atuaggcatcagga-tatcactacaa:~gc . 540 tg: tg ac :

ttacttgtga ggggtgcaaaggtaagggtaaggcgcaaccaaacagacagtgctgcgcac600 gcgcacacgt gtgtatgtgtgtgtgcacgtgcgtgtgcaatcgttataaaacatgagaaa660 caaaaccagg aaaaagtatgtctaaagggatcaatctgtgtgatcaaagataggagaata720 aaatgttaag tttaaggttagtttctttcctttcctttcctttcctttcctttcctttcc780 tttcctttcc ctttccctttccctttccctttcccttcctctttctttctttctttcttt840 ctttctttct ttctttctttctttctttctttctttctttctttctttctctctctctct900 ctctctctct ctctctctctctctctctctctctctctctctccctctctctctctctct960 ctctctctct ctctctctctctctctctctctctctctctctctctctctctctctccct1020 ccctccccccctctctctcttttgaagtagagtatcattgtatagccctggctggcctag1080 acctcactatggagatcaggttggctttgaactcacagatacctgcctgtctctgctttc1140 taggtgctgggattaaaggtgtgggccaccatgcctggaccatttctttctttatttttt1200 tttctcctcccttgtcttccccttcgtcctcatcttctcctcctcatctttcttcttttt1260 ttgagaaaaggtctctctatgcatccctggctgtcctggaactcactgtttagaccaggc1320 tgtccttgaactctcagagatccacctacctttgtatccttactgctggcattaagagca1380 ttcactaccaggtctgtctcttcatttttgatgaagactttcacactttactttaaactc1440 Seite 16 L- 0020-01-WO-01.ST25 gatacatata agcaaacgttttccctatttctagaagaatttgatgctggaacaaaacat1500 gacaatgtaa agatctgtaaaccatgacacctttttatgtggttgataaaatttatacac1560 ttggagaaaa tttgaaataaatgacttctatgattctaagtcaataaattttaaaaagtg1620 tttcgtatag aaagagaacaagctgaaagtcgggaagcaagggccagtgtttgcaatgat1680 atttgtaaag agcggaagcctctctgataaataacagttctgaaacagtctaccactcca1740 gaaatggata aacatcgggatgggaatgcaggccagtggtagagcacttagcacctactt1800 ttggatttga ttctagcaccctctgaaacaaaacaaaacaacccaaacaacagaaaacag1860 tttagtcaat gtaacacaaataaacgtgtttgtttagggtaaaattttacctgtaaatct1920 taaacctcaa cactggcaaagaatagccaa~aggatatgaggtgacagagtcctgtggcga198 0 gtagttaggcaggggtggcctgctcttgacggtgacgggttactgttaaaactgagctga2040 agaacccagcacagttttgaaaacctgagctgagctttttcttccttcgttgaactgaaa2100 tggcgaaactccatctttcaagtgtccactgcaagagagtaagtggtggggctttgggac2160 aactcataggagggagagcaggaaatggcaggtattactgtgtaataaggttcgaaggag2220 gaaaagggatgggaactatctggcaagtgctgggaaagggagagagagcatctgcggatg2280 agggagggagggaccacctttgtgggctgcaactcagactgggagggtttagaaaagaga2340 ggcttttctgaattttttctttattttaggtgaccatgtattctctcaaaggaaactcga2400 gagatgcattgtaggatcttgatattttgtataatgcagttaaaaaaaaaattcttatag2460 gtaatttacctatagtagggtagagagttgtcttaatgtgataatgatactccatttaat2520 ctccagacgttataaagaaagacccagcctcaggatgtcaactgaactatactaactata2580 aagaattatagaattagctaactttccagataaatgtatatttaaaaaaatgtcgggaat2640 aaaaaaaaccctatttaattgaaatactctagttagcttgattactttctataaaggtaa2700 aatgttaataaaaatgagaagtaactaaacatatccatgtcaaactttgaccatctttgt2760 tgttaggctttttccggcgtagcattaccaagaatgcagtgtattcttgcaagaacggtg2820 gtcactgtgaaatggacatgtacatgcgcagaaaatgccaagagtgcagactgaagaagt2880 Seite 17 L-0020-01-WO-01.ST25 gtaaggcggt ggggatgttggcagaatgtaagtgccagtgtgttcgtggcttgcttttct2940 gaggggattt aacttttcccagccttaccttcctgacaaggaggacatgatagtggtggt3000 ggtttttcca gggcaagaatcgcccattgcactagggatgtgctgactgacccctgtgat3060 ttccacaaat gtgggaaactcaggtgcatcatttgtgttagtgaaggactgtattcacac3120 tctcctctat ataataaaaatgaatttaaaaattaataaaaaattaaaaatttccaacct3180 tcttgaggac ataaagcattcttcaagagttattttgaacatggccgtattcgtttgtat3240 atgcacacca cacacatatgtacagtaattgcgccacgtggatggcccattatcaattta3300 . agtag tgattccca taaatgt~tgccctgtt~cta to - ctaat~cttgaaa~tragtca3~a0. .
ttgLt t agtttcatct taattagatg ttctctgaga cctgaagagt agttaggcag ttaagagcat 3420 taggagagga cctgggcccg gtccccagct tccacgtggc ccctcacaat catttataac 3480 tcagttcctg agcatcccag gccctcttct gacctctgca ggtatgagtg ttgtgtgcat 3540 acacacatgc aagcaaaaca cacacataaa ataaaagaaa cctccatagt gatttaaaaa 3600 aggtattgta tgtttctgtt acttaaccta aaaatatata ctgccttgaa aagtgcaaag 3660 ttaaagttag tagttcccaa aaagaaagaa agaggaggag aaagaagaga aaaggaagtc 3720 ttccctttct gagaaggggt tctcagagtc ctaacacact acagtcttag gacaattgaa 3780 gggtaactgg atctctggac ccctgacatc ctcatatgta aagtccacta atagtctcac 3840 gagtgtcagg actcatctct gaatatcaac acattagcat atgagcagtg ggagatacaa 3900 ctcagtccaa agaaaccttt gaaaaagttg tcttttattt tattcattaa tttaatttgt 3960 attcaggatc tcactatgta gctgacctgg aacttgctat gtacgccagg ctggcatcaa 4020 actcagagat ccccctgcct ctgccctcca atttctggct caaaagttgt cttttaacct 4080 gtggcaggca atgcatgtga aagggaagtt ggagtcctct gaggaactct atgcataaaa 4140 tgagtcatcc tacacttaaa gaaacttaaa gaaattctgg caaggtgaag ctcgagagag 4200 ttggtgagca gtcacggtga ttggcgagca taagctggtc cgcaccctgc caccattgct 4260 Seite 18 L-0020-01-WO-01.ST25 tggatctgca cacttcatttacctttcagttgtcctaatactgccatgtgttagtgctct4320 gacggcccct cctcctcttccttccctcattctcctttccctttcctcctttcttttcct4380 cttcctcctc ttccccctcttcttcctccttttcctcctcttcgttctcttcttgtggtt4440 ttccttctca tggtcctgggggttgagttcaggtttctcacatcctagacaatctaccac4500 taagctacac ccatagactcctttcctactaaaaaacactgagttgccatggctggtatt4560 caacgtgaga tcatcttgtaacagtgtcttaagtagctggggttataggtgtttactacc4620 atgttcaata ctcaatatgcttaaaatggctttatttttttgtgtattctgttctctgac4680 aaataataaa ataatatagaggggttttaaggtttttgtttatacttcagctcctcatta4740..

gttttttatt ttataggtgtgtttttaaaaatactttgaaaacgacaggtatattaatag4800 ttggatactt ttagtagctg ctaaaagtac ttagagtagt ccagtccagt ccagtacata 4860 cactacagct cacaaccagc tatagtctca cttcaagggg ctatgacccc tctgtgctct 4920 gtgggcacct gcactcacct gtgcatttcc ccatacagac tcatgcaagt aattataaaa 4980 tatttaaaaa caaacaaaaa caatcctaaa ataaatatta tttgcctttg aaaaagaatt 5040 aaatgctttg tctgacatag gaaaaattga cttagcttat ttttaaaaat cctaagatag 5100 tcttttttaa ccttattaat aaaaccagtg ataatagtca tagataatgt aattctacta 5160 ttctatcaaa tgactttttc acttccaaga tgcaactaat gtgtaaatct cttatgtaaa 5220 aatctaaaca taatttggaa atgaaaaatt aattcttaat ctttgttata actattttta 5280 agcagacaag gtgtctatga agggaaaagc agaataggaa atttaataga tgcagacatg 5340 cacgagagat atctaggact atttactcac tgaagaccat tttaatcaaa ccctacagtg 5400 ttgttttccg agccaaaagt acttaatgtg aattctgtaa tgtcacaggt ttgctcacag 5460 agatccagtg taagtcaaag agacttcgca agaacttcaa gcacgggcct gccctgtacc 5520 ctgccatcca agtggaagat gaaggagcag acaccaaaca cgtgtcatcc agcaccagat 5580 ctgggaaagg ggcaagacgc ttcttccata cagtctcttg tgtctttatg aacagtgtgt 5640 aacaagcaca cattacaggc gccctctgtt tccttatagg ttcaggacaa catgactcta 5700 Seite 19 L-0020-02-WO-01.ST25 actcaagagg aacatcggct tctgaatacc atagtgactg ctcaccaaaa atccatgatt 5760 cccttgggag aaacaagcaa acttgtatgt atccattgct ggcaagaaat aatttgtaat 5820 gaatgatatt aggaaatgat agcaagaaca gtaaaaaaga aacaccttag taattatcag 5880 aaagacatac tatgaatcat ttttatttat ttattttttt tatggaccag ctgcaggagg 5940 gttccaaccc cgaactaagt tttctgagac tctcagaggt atcagtcctg cacatacaag 6000 ggctaatgaa gtttaccaag ggactcccag gtaaaattct gtttcattat tttttaagga 6060 tttatttatt tttatgttta tgtgttatgc atgttttgat tgcaattata tattcatatc 6220 atgtacacaa catattgtgg ~aggccagaag aga~tgttag ~tcccctaga actagagt,.ti .. ..6180 cctaccgatg gctgtgaatt accatgtggg cgttggaacc agagtctctg caagagcaag 6240 acaggctctt aactgctgat ctgtctttct agcccccaat ttttaaaaaa gattatttat 6300 ttatttattt atttatttat ttatttattt atttatctga gtacattgta gctgtacaga 6360 tggttgtgag ccttcatgtg gttgttgaga attgaatttt taggacctct gctcactctg 6420 gtcggccccg ctcgttctgg cccaaagatt tcataagtac actgtagctg tcttcaggtg 6480 caccagaaga gggcgttaga tctcgttacg gatggttgcg agccaccgtg tggttgctgg 6540 gacttgaact cagggctttt taggaagagt agtcagtgct cttacccact gagctatctc 6600 tccagcccca aattttcttt tttttaaagg gatagtatat caaacagaaa tttctagttg 6660 tggtactaga tctatcgctc agaggcagag cacctgtcta atgtgtgtga agccctgggt 6720 tttctagcaa tgttgcttct aactaaaaaa aaaaaaaatg tttatttgtc tttgtcaatt 6780 aatcagatga gtgacaatct aaatatttac aaaaattatt ataatgatgt gggctttctt 6840 agatgaaaca agaaacaaag aaggtttcct ctataatact aaacaagtca cagtttgacc 6900 aaaacttata tataactcag ctttattata tgcttagagg tacagaaact ctttacaaaa 6960 ttacattctt caaggctaat gcagctatgt tgatttgtcc ttgctgtaag aaagtgtaac 7020 ctagtgctga taaaagtgcc ccaaaataga aaaaaagaat tttattcaga tttcaattta 7080 Seite 20 L-0020-01-WO-01.ST25 tatactattg ttattcccca gaagcactta gggttaataa gtaaatattt ttaaacattt 7140 aatttatgaa cgttctcttt tgattatctg tttctttatg ctttatttat ttatttattt 7200 atttattttt aaggatttga aaatttaacc actgaggatc aggctgcatt acagaaggcg 7260 tcaaaaactg aagtgatgtt ccttcatgta gcccagcttt atggtgggaa agactcaacc 7320 tctggaagta aaaagaaaaa cagaatttta ttcaacttca agtacacaca gacagacaga 7380 cagacagaca gacacacaca cacacacaca cacacacaca cacacacacg cacgcacgca 7440 cgcacacact tttaaacact gttttttgga aatagactct catgtagtcc aggctatgta 7500 gaactcagta tgtagctaag gaggatctta aagccctgct gtgtctgctc ttacctgcca 7560 accatcagcc tagagcctct atgcctgcct tctatggtgc tggggacaga agccagggtc 7620 tgtgctcagt aagccagaac tcctaactcc taacatcatc agcctttcat ttatcttttc 7680 attttatttt accagttagt gtaggaacaa atgtctcata gtagctgttt ataaatagtt 7740 tcacattcat ctagcatttt ctctctaccg tgtaattctt ttaaaaattg tatttcttat 7800 tcttgtgaga actgtataca tgtcctaagt gttttttttt ttaaatctta ctcacctcac 7860 ttccttccca ccagctcctt tagaatcccc cacacacctc tctcctaact tcatgtcctt 7920 ccctgctcct tctttttcaa aataacccat tgactctaaa tagtgctgct catatacacg 7980 ggggtggggg tagggtcatc cactgaagca tggccaactt agagggctca gttccctaaa 8040 gagactggat tgtccttccc ctagcagctg ccaacagctt ctcagctgtg ggtggggctt 8100 ctgtgggtgg ggcttctgtg ctccttagac aggctcttcc aatgaggagg acttttatgg 8160 gtgatcaaac ataaaggcgt tggtgattct aagacacaaa gcagctctgg ggacggccgc 8220 atgcttgctt tgaaactcat aaccacgggc tactgtttgt ggaacgtctc tttactgggg 8280 tgaagcccta ttaggttagt gttgaaccaa ctatgcagga cctcacttca ataaaaaaaa 8340 agcaagtatt ttttaaaaaa tcaataaacc atttgaatag agcaggaaag ccatcattgt 8400 ttagtgtgtg aatgcaagag tccaatctgt aaagtcgcat gttggttgag gacatgggaa 8460 gaggcaatag gacaaaatca aaaccaaaag ccaacaatgc tagctgcctt cactagctgt 8520 Sei to 21 L-0020-01-WO-01.ST25 actcgttacagtgcacagccttttaaaatatccttatttattgattgttctctgcaaaca 8580 .

ctagaccagaaggctctaaagggaggcccttgatgacctggtccatgattgcttgagtag 8640 tgtttgcttaaaagtctccacagcctctggtgcactgagtggttcgtggagggtgtgtgt 8700 gtttaagaaagccattcttagtttctatttcattatgcaaaactcattttggagggtctt 8760 ctgtacaatcttttttttttttttacaaaatatgaactaatatctgttttgtgttccagt 8820 aacaaaaatgagaatgatatcattttaggagtttttttttcccccacataatagttatgc 8880 tggatactgaaaagatactgaatttactatttatataatgattaagaaagactatcaaca 8940 tt tgtgtttctcttttgctai~gt~ctatgagaccagcaaagcc~tcag- ctggguc~ct?000: _ ..
tt ~ ..

agaggtgcat aatcctagcg ctgatgaaag tgttcattct ccggaaaact ttctcaagga 9060 aggctaccct tcggctcctc taactggtaa cactgtgcag taggaagcat caccttgggt 9120 ggacacgctt ccttttctcc ccttttcatt gcactacaaa gagaaggcca tgggctttct 9180 cacagctcac aggcgtctct gtggtggtga aagcatttat agacacagcc aatacagttg 9240 ttagtgttgg caggtcatca gtgagaattc tatttgtaac cattttctta ctttggcttc 9300 atcatggcaa ttctttccct atttaattgc gaagccattt ggggcctctg aattattctt 9360 aggatgatga tgtaatctac agccaacatc ccagtatcaa ccacttttat gggtagaagg 9420 gtaagcctta ctgtagataa tagagataca cggagagtgg cttctgtagg ctaaaggctc 9480 tcagttccac agcctcctcc ttggtccacg tgatgacaac ctctctttgt tctggtcctt 9540 ttcagtagca cacctaatgc gcccactctt gaggttcagg cctgggcttc aagggcacaa 9600 caaaggggat tgtctgtgcc ggccatcaca tcggtagcca tcttgaaccc cactctcttt 9660 tcaattttct tacactgttt gagagaagaa aagacaatca caatgtgttt gcttctgtgc 9720 tccattccga tgaaaattca cttatattaa tcttttcaga tattactaaa gaatttattg 9780 cctcactatc ttacttctac agaagaatga gtgaacttca tgtatcggat actgaatatg 9840 ctctgcttac ggcgacaaca gtgcttttct caggtacaga ctgagaagta aggctcggag 9900 Seite 22 L-0020-01-WO-01.ST25 tgatctctgg acaggcaatc actaagttct ttttatcgaa ctatgaactg aagttgaatg 9960 taccactctc atcccatcat gctttgctct ctatgtgcat tcctttaagc ttaccactgg 10020 gttacatctt tcttgcatct taaagaaatg ctttcttgcc ctcatagccc ctcagatata 10080 cgctcccagt ccctgataga gagaggttct cagggagtta gctatattct agtttccacc 10140 ttactcatgc tcactcctga gttcaccaca gtcacatttc actccaccct ctccagtcca 10200 ctacaacctg tcaggatctt caggacatgc tatcactgac tcaactggtc aatgctctgt 10260 cttcatctta cttgccccac tggtgtattt tgtagagagt atcataagca tctgaaagaa 10320 aagaagggga tatgttgttt cctgtgagcc cttcagtgga attttgaagc acggtgaaga 10380 tgtctgatgt gttaggagaa tttctcatgc ttctttatgt cagttcacag tcttcttgac 10440 tatgcgttac aaaccggacc caggttgaag tgagacagtt acttagcatg ggtaacccat 10500 tgctcaagcc agcgacgtct tcctagggga ccattgtcct gtgtccttca ccgattcggc 10560 catgttt,gtg ttcttttata ttctaacttg ctgtgatatc agggttcgtg aatgttcact 10620 aggtatgtgc atctgaagtt gaatcacata ggctccagag cagctccatt atattgtcca 10680 cttttattat gctttattcc aagaagtttt ggaagcaact gtgtgacaaa caggataaag 10740 gatgtagaag gctggaggga cagcagtgtt ggctatcatc tgaatctgaa tatttggaaa 10800 atcagctcag actgttacct agggacaggt cactctggtt ggaccataaa actcttcttt 10860 ctttcagttc agtgtaggga attaagctac aaattctgga tggtgcagta ccttagagcc 10920 ctggtgttct ggctgtgtga ggctttcccc atctctcagg agtgggactg ataacgcctg 10980 tggccatcat cctttatcct tcttggtggg tcttttaaaa caaggactgt gtcatgctct 11040 ctacatttaa gaatttcaat ggggccacag gcgcccctag attataaagt tctcataaag 11100 atatgttcat cacatccctt cagctgatct ttgggtttgc agggctaata tttgattccc 11160 aacaatttac cttttcataa tcttgccatt tctacttact gtttagatgg aaaagagtaa 11220 gttctccttg tagactgcag tccacctaac aacttgcctc cagccttgtt aacagtacca 11280 caagcagaac taaaatcatg caaagaattg cagaagcatg gcagtctcag gaagtttcca 1134D
Seite 23 L-0020-01-WO-01.ST25 ctccaaaaaa accccaaaac aaaaacaaaa acaaaaacaa aaacaaaaaa ccccacccac 11400 tcactaactc acataggtgg tttgtttcct tttgttggac atttacaatt ctatctatct 11460 atctatctat ctatctatct atctatctat ctatctatct atctatctat ctatctatcc 11520 tactatctat ctatctatct atctatctat ctatctatct atctatctat ctatctatct 11580 atctacctac ctacctatct atctatctat ctatctatct atctatctat cctactatct 11640 atctatctat ctatctatct atctatctat ctatctatct atccatccac tgtgtgtgtg 11700 tacatgtcat gcttgtggag gtcagaggac aacctatggg aattgtttct ctctttccac 11760 c~tgtgtgtt ctggggttc-a g~tagtcagg Ctgggtgata-gcatcttcec tgctgggctaw.11820. . -, tcccactggc agctttgttg tttcacggaa tctctggaac atcaccctct taatttctct 11880 gttcattggc tgttctttct gtgccttctt tattctgtca tcttcgtctc tcacaatgac 11940 aaatatttaa gttgtcctag gacccagttt tcttttaaat tattttatga ttttatgcac 12000 atgtttcgcc tgcatctatg caccatggtg tgcctcacgc tgctgagttc agaagatatc 12060 aggtcccttg ggacttgact tacagaggtt gtgacaaggc ccagttttct tctgcactgg 12120 cttccttcct gattccatcc taactcttgg cattaaaggc ctgtgtggat tcgatactcc 12180 cagacgtatc tgtgtgctcc cccagaatgt attaaatagc ctcctcaagt tttccatgta 12240 accgtcaaga aggtgtatca aatttaaaat gcccaaagct ggactcttga cacctttata 12300 ccttagcttt tggtattcct cccccatctg gaccagacca tagcttcgtc ttccaaattg 12360 catgtgcaac tttctttttt aatccttagt ttcttttctt tctcttatat tcatatcaca 12420 atgcacccgt tctgtttatt caaccctaag atatacccag cacttaaccg cttcctaccg 12480 tgtatagctt gggcagcgta ggtgaatcca cttttctaac tggattttct gactctatgc 12540 tttatagact ctctctgtat tagtacacag aggggttccc tggaaatata agtcagatga 12600 tggtatgcct ctggaactgt cgatggcttc ctagtttgca tagattaaaa ccaaaggtcc 12660 gggggggggg ggctggagag atggctcagt ggttaagagc agtgactgct cttccaaagg 12720 Seite 24 L-0020-01-WO-01.ST25 tcctgagttc aattcccagc aaccatatgg tggctcacaa ccatctgtaa tgagatctga 12780 ctccatcttc tggagtgtct gaagacagct acagtgtact cacatataat aaaaataaat 12840 aaatcttaaa aaaaaaaacc aaaggtcctt ctattggtct agtttgtctt gctccttttt 12900 catcctcgcc catccacatt gctttgacac aggtttccct tgatctcgca ggtttctgct 12960 acatgctctg ccatttttga tgcatctttt gccccctttc cttggatgcc cacatgatgc 13020 catcttacac tgttttgcca ttttatctac ttgttttttt cttttacctt tttgggtttg 13080 ggttaaatgt tgttgtatcc ctgattaatc tttacaaata gctaccaccc ccccccaaga 13140 gtcttattta tttctgatta ctttttttaa tggcacatga ttattttgtt tttgcttctt 13200 taaaaaaacc tttttcattc attgataaaa agcacttact ttccttgatg tgtatgtctc 13260 ctttgtctaa atgtgcaccc cttgtgtgct gtgagtccac aagaaggtgt catatcccct 13320 ggaactggag ttacagatgg ccgtgagcca ccatgtgggt gtggaaatca aaccccagta 13380 cttgggaaga gcagccagtg ctcttagctg ttgataggtc tttccagact gtttttactt 13440 ctgaagatgt gtgtaagtct ctccctctcc acttcttcca cattaagaat ataaggacaa 13500 taaagggaca caatcttgcc tgtgagtttc attgtggtat agttagcaag acgactggat 13560 gaatgaaggc tgcctaagcc ttcggaactt cctggttagc tcatttctta cgtaggaacc 13620 caagaagtct cacgctttac ctctgttgac tgtagagagg ttacttacta ctattttaaa 13680 gtttcaggca agcgtttatg gctgttatat ccccaacctt actcaccgat ccctttcttc 13740 ccatgtgact ttatggaaat tacttaactc cctacatttc agggagctaa tctgaaaaaa 13800 atgtgataaa aatagaatgt agctcaatag aatatagtga agattaagta aagtcatgaa 13860 caaaaagaat ttagttagaa aacctaatgt gtattagggt tctgcaagta cttatgattt 13920 tataactctg ttaataactc attgttttaa gtgcattcat tattcccacc actttgaatc 13980 attattaaga taaatatgat gtttttcaga tcgtccatgc cttaaaaata agcagcatat 14040 agaaaaccta caagaaccag tcctgcaact tttgtttaag ttttcaaaaa tgtaccatcc 14100 agaagaccca cagcatttcg cccacctcat agggaggctt actgaactga gaactctgag 14160 Seite 25 L-0020-01-WO-01.ST25 tcacagccac tctgaaatcc ttcgcatgtg gaaaacaaag gaccccaggt tggtgatgtt 14220 attctctgag aaatgggatc tgcactcatt ttcctgaaaa tttacagtgg tgagacttgc 14280 cttaaaattt caaaatcaaa attggatgaa tgatttgaaa aaaatgtgat aactcaaaat 14340 cattgagtta tcacacacca ccttaaaatg gagatccaaa aggctcatca gggtttgtgg 14400 aactttcaac tcttagaaga tgaacactcc aggattcatc tcaagattgg atgcaaagga 14460 aagaaaaatg gagccatgtg gtggcatagg ctctgtagtt gagagacaga gacaagagga 14520 ccccatgttt aaggcagtcc tgggctatgt agtgagacct catctcagaa gaaaaaagag 14580 _ .. ua~aactgat ggcatatt atgtctceaa tcetcaccta a:~gaaactaa t~ctTTtgtt,g ~~5~:~-.
ttgttgttta gctgaaacac ataatgtgta aatgtgaaca caggtaggtt atggttagga 14700 taaggcagtt tgtatgcttc actggttaag gacacatgga tggtgtagca gctaatgctt 14760 agaacctctt cttcatgaat gattaacgag gaacaagcct tttcaaattg tatctgcttt 14820 atatttttaa taagtatgtg ataattgtac agaatagtca atattgtaca gaatatttca 14880 atatgggaat ttcatgtgta tatattagat atttctggcg tatttgccac ccaccctgtg 14940 atgcccacac tgccaatgcc cttctcatcc ccctaacatt tcccttcctc tgccttgccc 15000 taaaacacca gattcatacg tgcaagaaaa cattttttaa acaaatttta ttagatattt 15060 tcttcattta caattcaaat gctatcccaa aagtccctta ttccctcccc ttatcctgct 15120 ctccaaccca cccacttctg cttcctggcc ctggtattcc cctgtattgg ggtatataat 15180 ctttgcaaga ccaagggcct ctcctcccaa cgatggtcca ctaggccatc tgcaagaaaa 15240 catttgattc cagtctgagt ctgttttatt tcctttgagt atgactatct cgtcaatatc 15300 tgcaaatgac ataatttcat tcctcttaag gatggaaagg actggattgt gtatgtatat 15360 ctgtatatat tattttattt atccacctgc tgatgacacc tagtttgatt ctgtatcttg 15420 gccactgtga atcgtgctat aataaaccta tgtaggtttt tagattttat gtggctcttg 15480 attctttcag gtgtactccc agaagtggta tggtttatat ggtagctcta tttttagatt 15540 Seite 26 L-0020-01-WO-01.ST25 tgaagggaac tatgtagtga ttttcacagt gattaccttc ttctcaatag tgtataaaga 15600 ttaatttgcc ttcaaaattc ttgccaattt ttgtttttcc ctttcctttt ctttttaatt 15660 aaaaaagtgt gtgtgtgtgt gcgtgcatgc caaagaacat tgtggacatc ggaggacagc 15720 tctgtggaat acattctctc cttccacctt tatgggtggt ctcctctgac cccctctgcc 15780 atggcctgca tggcttggtt ctggaagcct gattggggtg atgagcgaat gaagaaatga 15840 cagacatgaa gacacaagta cagaaaagct gagattgggt ggtgtctaag tttctttttt 15900 attgctataa agagacacca ggaccttatg aaagaaagca ttaaattggg ggcttgctta 15960 cagtcttaaa gattgcgtcc atgatgatca tggcaggtag catggcacca ggcatgcact 1602 0 ggaacagtat ctgaagagct tacattctga tcggcaagtt ggaggcaggg ggtgggggga 16080 gaagaaagtg gctgatgggg cctgggacag gatttggaaa tctcaaagcc catccctagt 16140 gacacacctc ctccaacgag gccacaccca gtcctcctca aataggtcca ccaagtaggg 16200 <210> 21 <211> 16200 <212> DNA
<213> Mus musculus <400> 21 ccctacttgg tggacctatt tgaggaggac tgggtgtggc ctcgttggag gaggtgtgtc 60 actagggatg ggctttgaga tttccaaatc ctgtcccagg ccccatcagc cactttcttc 120 tccccccacc ccctgcctcc aacttgccga tcagaatgta agctcttcag atactgttcc 180 agtgcatgcctggtgccatgctacctgccatgatcatcatggacgcaatctttaagactg240 taagcaagcccccaatttaatgctttctttcataaggtcctggtgtctctttatagcaat300 aaaaaagaaacttagacaccacccaatctcagcttttctgtacttgtgtcttcatgtctg360 tcatttcttcattcgctcatcaccccdatcaggcttccagaaccaagccatgcaggccat420 ggcagagggggtcagaggagaccacccataaaggtggaaggagagaatgtattccacaga480 gctgtcctccgatgtccacaatgttctttggcatgcacgcacacacacacacactttttt540 Sei to 27 L-0020-01-WO-01.ST25 aattaaaaag aaaaggaaag ggaaaaacaa aaattggcaa gaattttgaa ggcaaattaa 600 tctttataca ctattgagaa gaaggtaatc actgtgaaaa tcactacata gttcccttca 660 aatctaaaaa tagagctacc atataaacca taccacttct gggagtacac ctgaaagaat 720 caagagccac ataaaatcta aaaacctaca taggtttatt atagcacgat tcacagtggc 780 caagatacag aatcaaacta ggtgtcatca gcaggtggat aaataaaata atatatacag 840 atatacatac acaatccagt cctttccatc cttaagagga atgaaattat gtcatttgca 900 gatattgacg agatagtcat actcaaagga aataaaacag actcagactg gaatcaaatg 960 ttttcttgca gatggcctag tggaccatcg ttgggaggag aggcccttgg tcttgcaaag 1020 attatatacc ccaatacagg ggaataccag ggccaggaag cagaagtggg tgggttggag 1080 agcaggataa ggggagggaa taagggactt ttgggatagc atttgaattg taaatgaaga 1140 aaatatctaa taaaatttgt ttaaaaaatg ttttcttgca cgtatgaatc tggtgtttta 1200 gggcaaggca gaggaaggga aatgttaggg ggatgagaag ggcattggca gtgtgggcat 1260 cacagggtgg gtggcaaata cgccagaaat atctaatata tacacatgaa attcccatat 1320 tgaaatattc tgtacaatat tgactattct gtacaattat cacatactta ttaaaaatat 1380 aaagcagata caatttgaaa aggcttgttc ctcgttaatc attcatgaag aagaggttct 1440 aagcattagc tgctacacca tccatgtgtc cttaaccagt gaagcataca aactgcctta 1500 tcctaaccat aacctacctg tgttcacatt tacacattat gtgtttcagc taaacaacaa 1560 caacaaaaaa ttagtttctt taggtgagga ttggagacat aatgatggcc atcagttctt 1620 ctcttttttc ttctgagatg aggtctcact acatagccca ggactgcctt aaacatgggg 1680 tcctcttgtc tctgtctctc aactacagag cctatgccac cacatggctc catttttctt 1740 tcctttgcat ccaatcttga gatgaatcct ggagtgttca tcttctaaga gttgaaagtt 1800 ccacaaaccc tgatgagcct tttggatctc cattttaagg tggtgtgtga taactcaatg 1860 attttgagtt atcacatttt tttcaaatca ttcatccaat tttgattttg aaattttaag 1920 gcaagtctca ccactgtaaa ttttcaggaa aatgagtgca gatcccattt ctcagagaat 1980 Seite 28 L-0020-01-WO-01.ST25 aacatcacca acctggggtc ctttgttttc cacatgcgaa ggatttcaga gtggctgtga 2040 ctcagagttc tcagttcagt aagcctccct atgaggtggg cgaaatgctg tgggtcttct 2100 ggatggtaca tttttgaaaa cttaaacaaa agttgcagga ctggttcttg taggttttct 2160 atatgctgct tatttttaag gcatggacga tctgaaaaac atcatattta tcttaataat 2220 gattcaaagt ggtgggaata atgaatgcac ttaaaacaat gagttattaa cagagttata 2280 aaatcataag tacttgcaga accctaatac acattaggtt ttctaactaa attctttttg 2340 ttcatgactt tacttaatct tcactatatt ctattgagct acattctatt tttatcacat 2400 ttttttca~a tta~ctccct gaaatgtagg gag~taagta atttccataa agtc~catgg 2450 _ .
gaagaaaggg atcggtgagt aaggttgggg atataacagc cataaacgct tgcctgaaac 2520 tttaaaatag tagtaagtaa cctctctaca gtcaacagag gtaaagcgtg agacttcttg 2580 ggttcctacg taagaaatga gctaaccagg aagttccgaa ggcttaggca gccttcattc 2640 atccagtcgt cttgctaact ataccacaat gaaactcaca ggcaagattg tgtcccttta 2700 ttgtccttat attcttaatg tggaagaagt ggagagggag agacttacac acatcttcag 2760 aagtaaaaac agtctggaaa gacctatcaa cagctaagag cactggctgc tcttcccaag 2820 tactggggtt tgatttccac acccacatgg tggctcacgg ccatctgtaa ctccagttcc 2880 aggggatatg acaccttctt gtggactcac agcacacaag gggtgcacat ttagacaaag 2940 gagacataca catcaaggaa agtaagtgct ttttatcaat gaatgaaaaa ggttttttta 3000 aagaagcaaa aacaaaataa tcatgtgcca ttaaaaaaag taatcagaaa taaataagac 3060 tcttgggggg gggtggtagc tatttgtaaa gattaatcag ggatacaaca acatttaacc 3120 caaacccaaa aaggtaaaag aaaaaaacaa gtagataaaa tggcaaaaca gtgtaagatg 3180 gcatcatgtg ggcatccaag gaaagggggc aaaagatgca tcaaaaatgg cagagcatgt 3240 agcagaaacc tgcgagatca agggaaacct gtgtcaaagc aatgtggatg ggcgaggatg 3300 aaaaaggagc aagacaaact agaccaatag aaggaccttt ggtttttttt tttaagattt 3360 Seite 29 L-0020-Ol-WO-01.ST25 atttattttt attatatgtgagtacactgtagctgtcttcagacactccagaagatggag3420 tcagatctca ttacagatggttgtgagccaccatatggttgctgggaattgaactcagga3480 cctttggaag agcagtcactgctcttaaccactgagccatctctccagcccccccccccc3540 ggacctttgg ttttaatctatgcaaactaggaagccatcgacagttccagaggcatacca3600 tcatctgact tatatttccagggaacccctctgtgtactaatacagagagagtctataaa3660 gcatagagtc agaaaatccagttagaaaagtggattcacctacgctgcccaagctataca3720 cggtaggaag cggttaagtgctgggtatatcttagggttgaataaacagaacgggtgcat3780 tgtgatatga atataagagaaagaaaagaaactaaggattaaaaaagaaagttgcacatg3840 caatttggaa gacgaagctatggtctggtccagatgggggaggaataccaaaagctaagg3900 tataaaggtg tcaagagtccagctttgggcattttaaatttgatacaccttcttgacggt3960 tacatggaaaacttgaggaggctatttaatacattctgggggagcacacagat~cgtctg4020 ggagtatcgaatccacacaggcctttaatgccaagagttaggatggaatcag~~aggaag4080 ccagtgcagaagaaaactgggccttgtcacaacctctgtaagtcaagtcccaagggacct4140 gatatcttctgaactcagcagcgtgaggcacaccatggtgcatagatgcaggcgaaacat4200 gtgcataaaatcataaaataatttaaaagaaaactgggtcctaggacaacttaaatattt4260 gtcattgtgagagacgaagatgacagaataaagaaggcacagaaagaacagccaatgaac4320 agagaaattaagagggtgatgttccagagattccgtgaaacaacaaagctgccagtggga4380 tagcccagcagggaagatgctatcacccagcctgactacctgaaccccagaacacacatg4440 gtggaaagagagaaacaattcccataggttgtcctctgacctccacaagcatgacatgta4500 cacacacacagtggatggatagatagatagatagatagatagatagatagatagatagat4560 agatagtaggatagatagatagatagatagatagatagatagataggtaggtaggtagat4620 agatagatagatagatagatagatagatagatagatagatagatagatagatagatagta4680 ggatagatagatagatagatagatagatagatagatagatagatagatagatagatagat4740 agatagatagaattgtaaatgtccaacaaaaggaaacaaaccacctatgtgagttagtga4800 Seite 30 L-0020-Ol-WO-01.ST25 gtgggtgggg ttttttgttt ttgtttttgt ttttgttttt gttttggggt ttttttggag 4860 tggaaacttc ctgagactgc catgcttctg caattctttg catgatttta gttctgcttg 4920 tggtactgtt aacaaggctg gaggcaagtt gttaggtgga ctgcagtcta caaggagaac 4980 ttactctttt ccatctaaac agtaagtaga aatggcaaga ttatgaaaag gtaaattgtt 5040 gggaatcaaa tattagccct gcaaacccaa agatcagctg aagggatgtg atgaacatat 5100 ctttatgaga actttataat ctaggggcgc ctgtggcccc attgaaattc ttaaatgtag 5160 agagcatgac acagtccttg ttttaaaaga cccaccaaga aggataaagg atgatggcca 5220 ca~g~:gttat cagtcccact cctgagagat gg~gaaagcc t~acacagcc agaacaccag .5?$~_-, .
ggctctaagg tactgcacca tccagaattt gtagcttaat tccctacact gaactgaaag 5340 aaagaagagt tttatggtcc aaccagagtg acctgtccct aggtaacagt ctgagctgat 5400 tttccaaata ttcagattca gatgatagcc aacactgctg tccctccagc cttctacatc 5460 ctttatcctg tttgtcacac agttgcttcc aaaacttctt ggaataaagc ataataaaag 5520 tggacaatat aatggagctg ctctggagcc tatgtgattc aacttcagat gcacatacct 5580 agtgaacatt cacgaaccct gatatcacag caagttagaa tataaaagaa cacaaacatg 5640 gccgaatcgg tgaaggacac aggacaatgg tcccctagga agacgtcgct ggcttgagca 5700 atgggttacc catgctaagt aactgtctca cttcaacctg ggtccggttt gtaacgcata 5760 gtcaagaaga ctgtgaactg acataaagaa gcatgagaaa ttctcctaac acatcagaca 5820 tcttcaccgt gcttcaaaat tccactgaag ggctcacagg aaacaacata tccccttctt. 5880 ttctttcaga tgcttatgat actctctaca aaatacacca gtggggcaag taagatgaag 5940 acagagcatt gaccagttga gtcagtgata gcatgtcctg aagatcctga caggttgtag 6000 tggactggag agggtggagt gaaatgtgac tgtggtgaac tcaggagtga gcatgagtaa 6060 ggtggaaact agaatatagc taactccctg agaacctctc tctatcaggg actgggagcg 6120 tatatctgag gggctatgag ggcaagaaag catttcttta agatgcaaga aagatgtaac 6180 Seite 31 L-0020-01-WO-01.ST25 ccagtggtaa gcttaaagga atgcacatag agagcaaagc atgatgggat gagagtggta 6240 cattcaactt cagttcatag ttcgataaaa agaacttagt gattgcctgt ccagagatca 6300 ctccgagcct tacttctcag tctgtacctg agaaaagcac tgttgtcgcc gtaagcagag 6360 catattcagt atccgataca tgaagttcac tcattcttct gtagaagtaa gatagtgagg 6420 caataaattc tttagtaata tctgaaaaga ttaatataag tgaattttca tcggaatgga 6480 gcacagaagc aaacacattg tgattgtctt ttcttctctc aaacagtgta agaaaattga 6540 aaagagagtg gggttcaaga tggctaccga tgtgatggcc ggcacagaca atcccctttg 6600 ttgtgccctt gaagcccagg cctgaacctc aagagtgggc gcattaggtg tgctactgaa 6660 aaggaccaga acaaagagag gttgtcatca cgtgg~accaa ggaggaggct gtggaactga 6720 gagcctttag cctacagaag ccactctccg tgtatctcta ttatctacag taaggcttac 6780 ccttctaccc ataaaagtgg ttgatactgg gatgttggct gtagattaca tcatcatcct 6840 aagaataatt cagaggcccc aaatggcttc gcaattaaat agggaaagaa ttgccatgat 6900 gaagccaaag taagaaaatg gttacaaata gaattctcac tgatgacctg ccaacactaa 6960 caactgtatt ggctgtgtct ataaatgctt tcaccaccac agagacgcct gtgagctgtg 7020 agaaagccca tggccttctc tttgtagtgc aatgaaaagg ggagaaaagg aagcgtgtcc 7080 acccaaggtg atgcttccta ctgcacagtg ttaccagtta gaggagccga agggtagcct 7140 tccttgagaa agttttccgg agaatgaaca ctttcatcag cgctaggatt atgcacctct 7200 agtgtcccag ctgagggctt tgctggtctc atagtaccta gcaaaagaga aaaacacaaa 7260 tgttgatagt ctttcttaat cattatataa atagtaaatt cagtatcttt tcagtatcca 7320 gcataactat tatgtggggg aaaaaaaaac tcctaaaatg atatcattct catttttgtt 7380 actggaacac aaaacagata ttagttcata ttttgtaaaa aaaaaaaaaa gattgtacag 7440 aagaccctcc aaaatgagtt ttgcataatg aaatagaaac taagaatggc tttcttaaac 7500 acacacaccc tccacgaacc actcagtgca ccagaggctg tggagacttt taagcaaaca 7560 ctactcaagc aatcatggac caggtcatca agggcctccc tttagagcct tctggtctag 7620 Seite 32 L-0020-01-W0-01.ST25 tgtttgcaga gaacaatcaa taaataagga tattttaaaa ggctgtgcac tgtaacgagt 7680 acagctagtg aaggcagcta gcattgttgg cttttggttt tgattttgtc ctattgcctc 7740 ttcccatgtc ctcaaccaac atgcgacttt acagattgga ctcttgcatt cacacactaa 7800 acaatgatggctttcctgctctattcaaatggtttattgattttttaaaaaatacttgct7860 ttttttttattgaagtgaggtcctgcatagttggttcaacactaacctaatagggcttca7920 ccccagtaaagagacgttccacaaacagta.gcccgtggttatgagtttcaaagcaagcat7980 gcggccgtccccagagctgctttgtgtcttagaatcaccaacgcctttatgtttgatcac8040 ~~cat~aaaagtrctcctcatt~gga.agagcctg~cc~~a~jgga9c.ar~aga.agcc.cca~~c~ca.;_,8100 a~~ ,. ..

aagccccacccacagctgagaagctgttggcagctgctaggggaaggacaatccagtctc8160 tttagggaac tgagccctct aagttggcca tgcttcagtg gatgacccta cccccacccc 8220 cgtgtatatg agcagcacta tttagagtca atgggttatt ttgaaaaaga aggagcaggg 8280 aaggacatga agttaggaga gaggtgtgtg ggggattcta aaggagctgg tgggaaggaa 8340 gtgaggtgag taagatttaa aaaaaaaaac acttaggaca tgtatacagt tctcacaaga 8400 ataagaaata caatttttaa aagaattaca cggtagagag aaaatgctag atgaatgtga 8460 aactatttat aaacagctac tatgagacat ttgttcctac actaactggt aaaataaaat 8520 gaaaagataa atgaaaggct gatgatgtta ggagttagga gttctggctt actgagcaca 8580 gaccctggct tctgtcccca gcaccataga aggcaggcat agaggctcta ggctgatggt 8640 tggcaggtaa gagcagacac agcagggctt taagatcctc cttagctaca tactgagttc 8700 tacatagcct ggactacatg agagtctatt tccaaaaaac agtgtttaaa agtgtgtgcg 8760 tgcgtgcgtg cgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtc tgtctgtctg 8820 tctgtctgtc tgtgtgtact tgaagttgaa taaaattctg tttttctttt tacttccaga 8880 ggttgagtct ttcccaccat aaagctgggc tacatgaagg aacatcactt cagtttttga 8940 cgccttctgt aatgcagcct gatcctcagt ggttaaattt tcaaatcctt aaaaataaat 9000 Seite 33 L-0020-01-WO-01.ST25 aaataaataa ataaataaag cataaagaaa cagataatca aaagagaacg ttcataaatt 9060 aaatgtttaa aaatatttac ttattaaccc taagtgcttc tggggaataa caatagtata 9120 taaattgaaa tctgaataaa attctttttt tctattttgg ggcactttta tcagcactag 9180 gttacacttt cttacagcaa ggacaaatca acatagctgc attagccttg aagaatgtaa 9240 ttttgtaaag agtttctgta cctctaagca tataataaag ctgagttata tataagtttt 9300 ggtcaaactg tgacttgttt agtattatag aggaaacctt ctttgtttct tgtttcatct 9360 aagaaagccc acatcattat aataattttt gtaaatattt agattgtcac tcatctgatt 9420 aattgacaaa gacaaataaa catttttttt ttttttagtt agaagcaaca ttgctagaaa 9480 ~acccagggct~tcacacacat tagacaggtg~ctctgcctct gagcgataga tctagtacca ,9540 caactagaaa tttctgtttg atatactatc cctttaaaaa aaagaaaatt tggggctgga 9600 gagatagctc agtgggtaag agcactgact actcttccta aaaagccctg agttcaagtc 9660 ccagcaacca cacggtggct cgcaaccatc cgtaacgaga tctaacgccc tcttctggtg 9720 cacctgaaga cagctacagt gtacttatga aatctttggg ccagaacgag cggggccgac 9780 cagagtgagc agaggtccta aaaattcaat tctcaacaac cacatgaagg ctcacaacca 9840 tctgtacagc tacaatgtac tcagataaat aaataaataa ataaataaat aaataaataa 9900 ataaataatc ttttttaaaa attgggggct agaaagacag atcagcagtt aagagcctgt 9960 cttgctcttg cagagactct ggttccaacg cccacatggt aattcacagc catcggtagg 10020 aaactctagt tctaggggac caaacactct cttctggcct ccacaatatg ttgtgtacat 10080 gatatgaata tataattgca atcaaaacat gcataacaca taaacataaa aataaataaa 10140 tccttaaaaa ataatgaaac agaattttac ctgggagtcc cttggtaaac ttcattagcc 10200 cttgtatgtg caggactgat acctctgaga gtctcagaaa acttagttcg gggttggaac 10260 cctcctgcag ctggtccata aaaaaaataa ataaataaaa atgattcata gtatgtcttt 10320 ctgataatta ctaaggtgtt tcttttttac tgttcttgct atcatttcct aatatcattc 10380 attacaaatt atttcttgcc agcaatggat acatacaagt ttgcttgttt ctcccaaggg 10440 Seite 34 L-0020-Ol-WO-01.ST25 aatcatggat ttttggtgag cagtcactat ggtattcaga agccgatgtt cctcttgagt 10500 tagagtcatg ttgtcctgaa cctataagga aacagagggc gcctgtaatg tgtgcttgtt 10560 acacactgtt cataaagaca caagagactg tatggaagaa gcgtcttgcc cctttcccag 10620 atctggtgct ggatgacacg tgtttggtgt ctgctccttc atcttccact tggatggcag 10680 ggtacagggc aggcccgtgc ttgaagttct tgcgaagtct ctttgactta cactggatct 10740 ctgtgagcaa~ acctgtgaca ttacagaatt cacattaagt acttttggct cggaaaacaa 10800 cactgtaggg tttgattaaa atggtcttca gtgagtaaat agtcctagat atctctcgtg 10860 .. .. . ~~tgtctgca tetattaaat ttcc~attct. ;gcttttcc:ct.. tcataga,cac .ctt,gtctgct ~ln~~.~: - - '. .. ..
taaaaatagt tataacaaag attaagaatt aatttttcat ttccaaatta tgtttagatt 10980 tttacataag agatttacac attagttgca tcttggaagt gaaaaagtca tttgatagaa 11040 tagtagaatt acattatcta tgactattat cactggtttt attaataagg ttaaaaaaga 11100' ctatcttagg atttttaaaa ataagctaag tcaatttttc ctatgtcaga caaagcattt 11160 aattcttttt caaaggcaaa taatatttat tttaggattg tttttgtttg tttttaaata 11220 ttttataatt acttgcatga gtctgtatgg ggaaatgcac aggtgagtgc aggtgcccac 11280 agagcacaga ggggtcatag ccccttgaag tgagactata gctggttgtg agctgtagtg 11340 tatgtactgg actggactgg actactctaa gtacttttag cagctactaa aagtatccaa 11400 ctattaatat acctgtcgtt ttcaaagtat ttttaaaaac acacctataa aataaaaaac 11460 taatgaggag ctgaagtata aacaaaaacc ttaaaacccc tctatattat tttattattt 11520 gtcagagaac agaatacaca aaaaaataaa gccattttaa gcatattgag tattgaacat 11580 ggtagtaaac acctataacc ccagctactt aagacactgt tacaagatga tctcacgttg 11640 aataccagcc atggcaactc agtgtttttt agtaggaaag gagtctatgg gtgtagctta 11700 gtggtagatt gtctaggatg tgagaaacct gaactcaacc cccaggacca tgagaaggaa 11760 aaccacaaga agagaacgaa gaggaggaaa aggaggaaga agagggggaa gaggaggaag 11820 Seite 35 L-0020-Ol-WO-01.ST25 aggaaaagaa aggaggaaag ggaaaggaga atgagggaag gaagaggagg aggggccgtc 11880 agagcactaa cacatggcag tattaggaca actgaaaggt aaatgaagtg tgcagatcca 11940 agcaatggtg gcagggtgcg gaccagctta tgctcgccaa tcaccgtgac tgctcaccaa 12000 ctctctcgag cttcaccttg ccagaatttc tttaagtttc tttaagtgta ggatgactca 12060 ttttatgcat agagttcctc agaggactcc aacttccctt tcacatgcat tgcctgccac 12120 aggttaaaag acaacttttg agccagaaat tggagggcag aggcaggggg atctctgagt 12180 ttgatgccag cctggcgtac atagcaagtt ccaggtcagc tacatagtga gatcctgaat 12240 acaaattaaa ttaatgaata aaataaaaga caactttttc aaaggtttct ttggactgag 12300 ttgtatctcc cactgctcat atgctaatgtngttgatattc agagatgagt cctgacactc 12360 gtgagactat tagtggactt tacatatgag gatgtcaggg gtccagagat ccagttaccc 12420 ttcaattgtc ctaagactgt agtgtgttag gactctgaga accccttctc agaaagggaa 12480 gacttccttt tctcttcttt ctcctcctct ttctttcttt ttgggaacta ctaactttaa 12540 ctttgcactt ttcaaggcag tatatatttt taggttaagt aacagaaaca tacaatacct 12600 tttttaaatc actatggagg tttcttttat tttatgtgtg tgttttgctt gcatgtgtgt 12660 atgcacacaa cactcatacc tgcagaggtc agaagagggc ctgggatgct caggaactga 12720 gttataaatg attgtgaggg gccacgtgga ~agctggggac cgggcccagg tcctctccta 12780 atgctcttaa ctgcctaact actcttcagg tctcagagaa catctaatta agatgaaact 12840 atgactgatt tcaaaattag agcaatatag aacagggcaa catttatggg aatcactact 12900 taaattgata atgggccatc cacgtggcgc aattactgta catatgtgtg tggtgtgcat 12960 atacaaacga atacggccat gttcaaaata actcttgaag aatgctttat gtcctcaaga 13020 aggttggaaa tttttaattt tttattaatt tttaaattca tttttattat atagaggaga 13080 gtgtgaatac agtccttcac taacacaaat gatgcacctg agtttcccac atttgtggaa 13140 atcacagggg tcagtcagca catccctagt gcaatgggcg attcttgccc tggaaaaacc 13200 accaccacta tcatgtcctc cttgtcagga aggtaaggct gggaaaagtt aaatcccctc 13260 Seite 36 L-0020-Ol-WO-01.ST25 agaaaagcaa gccacgaaca cactggcact tacattctgc caacatcccc accgccttac 13320 acttcttcag tctgcactct tggcattttc tgcgcatgta catgtccatt tcacagtgac 13380 caccgttctt gcaagaatac actgcattct tggtaatgct acgccggaaa aagcctaaca 13440 acaaagatgg tcaaagtttg acatggatat gtttagttac ttctcatttt tattaacatt 13500 ttacctttat agaaagtaat caagctaact agagtatttc aattaaatag ggtttttttt 13560 attcccgaca tttttttaaa tatacattta tctggaaagt tagctaattc tataattctt 13620 tatagttagt atagttcagt tgacatcctg aggctgggtc tttctttata acgtctggag 13680 .. - a~aaaat.gga-'gtatcattat cacatt?aga caracvtc~tcta..ccctactata ggGaaal:i:ac~ i:~7.40. ..
ctataagaat ttttttttta actgcattat acaaaatatc aagatcctac aatgcatctc 13800 tcgagtttcc tttgagagaa tacatggtca cctaaaataa agaaaaaatt cagaaaagcc 13860 tctcttttct aaaccctccc agtctgagtt gcagcccaca aaggtggtcc ctccctccct 13920 catccgcaga tgctctctct ccctttccca gcacttgcca gatagttccc atcccttttc 13980 ctccttcgaa ccttattaca cagtaatacc tgccatttcc tgctctccct cctatgagtt 14040 gtcccaaagc cccaccactt actctcttgc agtggacact tgaaagatgg agtttcgcca 14100 tttcagttca acgaaggaag aaaaagctca gctcaggttt tcaaaactgt gctgggttct 1416 0 tcagctcagt tttaacagta acccgtcacc gtcaagagca ggccacccct gcctaactac 14220 tcgccacagg actctgtcac ctcatatcct ttggctattc tttgccagtg ttgaggttta 14280 agatttacag gtaaaatttt accctaaaca aacacgttta tttgtgttac attgactaaa 14340 ctgttttctg ttgtttgggt tgttttgttt tgtttcagag ggtgctagaa tcaaatccaa 14400 aagtaggtgc taagtgctct accactggcc tgcattccca tcccgatgtt tatccatttc 14460 tggagtggta gactgtttca gaactgttat ttatcagaga ggcttccgct ctttacaaat 14520 atcattgcaa acactggccc ttgcttcccg actttcagct tgttctcttt ctatacgaaa 14580 cactttttaa aatttattga cttagaatca tagaagtcat ttatttcaaa ttttctccaa 14640 Seite 37 L-0020-01-WO-01.ST25 gtgtataaat tttatcaacc acataaaaag gtgtcatggt ttacagatct ttacattgtc 14700 atgttttgtt ccagcatcaa attcttctag aaatagggaa aacgtttgct tatatgtatc 14760 gagtttaaag taaagtgtga aagtcttcat caaaaatgaa gagacagacc tggtagtgaa 14820 tgctcttaat gccagcagta aggatacaaa ggtaggtgga tctctgagag ttcaaggaca 14880 gcctggtcta aacagtgagt tccaggacag ccagggatgc atagagagac cttttctcaa 14940 aaaaagaaga aagatgagga ggagaagatg aggacgaagg ggaagacaag ggaggagaaa 15000 aaaaaataaa gaaagaaatg gtccaggcat ggtggcccac acctttaatc ccagcaccta 15060 gaaagcagag acaggcaggt atctgtgagt tcaaagccaa cctgatctcc atagtgaggt 15120 ctaggccagc cagggctata caatgatact ctacttcaaa agagagagag ggggggaggg 15180 ~agggagagag agagagagag agagagagag agagagagag agagagagag agagagagag 15240 agagagagag agagagggag agagagagag agagagagag agagagagag agagagagag 15300 agagagagag agaaagaaag aaagaaagaa agaaagaaag aaagaaagaa agaaagaaag 15360 aaagaaagaa agaaagaaag aggaagggaa agggaaaggg aaagggaaag ggaaaggaaa 15420 ggaaaggaaa ggaaaggaaa ggaaaggaaa ggaaagaaac taaccttaaa cttaacattt 15480 tattctccta tctttgatca cacagattga tccctttaga catacttttt cctggttttg 15540 tttctcatgt tttataacga ttgcacacgc acgtgcacac acacatacac acgtgtgcgc 15600 gtgcgcagca ctgtctgttt ggttgcgcct tacccttacc tttgcacccc tcacaagtaa 15660 gtgcattgta gtgatatcct gatgccttat caccgcagac catgcagagc atgtcatctt 15720 gacccttcca gcgtaaagaa caagttgctc tgcacctttg tgccccaggg aattcatctt 15780 catcatcgtg aacaaccaca taagtggact gaccagattc tcgagcgtcg agtccacacc 15840 aactgcctcc actgtactgt ggatccaagc catgtgtgtt gaaatgactt tgtaaagatg 15900 gggactgtaa cgctggagga aactgagcgg tagaatattg acaatagggt ggttcttgga 15960 aatctgtatc acacagctga taatggaatt gttctggcaa aatatctggt tttattaaca 16020 taaaaaagaa ~caaatcataa aaggtcaaaa aagttcataa ttgttttaaa cctcaaaatg 16080 Seite 38 L-0020-01-WO-Ol.ST25 ttttatactc aacagaaagt tagttcaaca catctattcc atctcaggac ttgccgtcca 16140 gcacaatagg aacacagtaa atggaactaa caatacctaa taaatgctat gaccccaaag 16200 <210>22 <211>1428 <212>DNA

<213>Mus musculus <400> 22 atgttaataa aaccagatat tttgccagaa caattccatt atcagctgtg tgatacagat 60 ttccaagaac caccctattg tcaatattct accgctcagt ttcctccagc gttacagtcc 120 ccatctttac aaagtcattt caacacacat ggcttggatc cacagtacag tggaggcagt 180 tggtgtggac tcgacgctcg agaatctggt cagtccactt atgtggttgt tcacgatgat 240 gaagatgaat tccctggggc acaaaggtgc agagcaactt gttctttacg ctggaagggt 300 caagatgaca tgctctgcat ggtctgcggt gataaggcat caggatatca ctacaatgca 360 cttacttgtgaggggtgcaaaggctttttccggcgtagcattaccaagaatgcagtgtat 420 tcttgcaagaacggtggtcactgtgaaatggacatgtacatgcgcagaaaatgccaagag 480 tgcagactgaagaagtgtaaggcggtggggatgttggcagaatgtttgctcacagagatc 540 cagtgtaagtcaaagagacttcgcaagaacttcaagcacgggcctgccctgtaccctgcc 600 atccaagtggaagatgaaggagcagacaccaaacacgtgtcatccagcaccagatctggg 660 aaaggggttc aggacaacat gactctaact caagaggaac atcggcttct gaataccata 720 gtgactgctc accaaaaatc catgattccc ttgggagaaa caagcaaact tctgcaggag 780 ggttccaacc ccgaactaag ttttctgaga ctctcagagg tatcagtcct gcacatacaa 840 gggctaatga agtttaccaa gggactccca ggatttgaaa atttaaccac tgaggatcag 900 gctgcattac agaaggcgtc aaaaactgaa gtgatgttcc ttcatgtagc ccagctttat 960 ggtgggaaag actcaacctc tggaagtact atgagaccag caaagccctc agctgggaca 1020 ctagaggtgc ataatcctag cgctgatgaa agtgttcatt ctccggaaaa ctttctcaag 1080 Seite 39 L-0020-01-WO-01.ST25 gaaggctacc cttcggctcc tctaactgga agaatgagtg aacttcatgt atcggatact 1140 gaatatgctc tgcttacggc gacaacagtg cttttctcag atcgtccatg ccttaaaaat 1200 aagcagcata tagaaaacct acaagaacca gtcctgcaac ttttgtttaa gttttcaaaa 1260 atgtaccatc cagaagaccc acagcatttc gcccacctca tagggaggct tactgaactg 1320 agaactctga gtcacagcca ctctgaaatc cttcgcatgt ggaaaacaaa ggaccccagg 1380 ttggtgatgt tattctctga gaaatgggat ctgcactcat tttcctga 1428 <210> 23 . . . , ,.

<212> DNA

<213> Mus musculus <400> 23 tcaggaaaat gagtgcagat cccatttctc agagaataac atcaccaacc tggggtcctt 60 tgttttccac atgcgaagga tttcagagtg gctgtgactc agagttctca gttcagtaag 120 cctccctatg aggtgggcga aatgctgtgg gtcttctgga tggtacattt ttgaaaactt 180 aaacaaaagt tgcaggactg gttcttgtag gttttctata tgctgcttat ttttaaggca 240 tggacgatct gagaaaagca ctgttgtcgc cgtaagcaga gcatattcag tatccgatac 300 atgaagttca ctcattcttc cagttagagg agccgaaggg tagccttcct tgagaaagtt 360 ttccggagaa tgaacacttt catcagcgct aggattatgc acctctagtg tcccagctga 420 gggctttgct ggtctcatag tacttccaga ggttgagtct ttcccaccat aaagctgggc 480 tacatgaagg aacatcactt cagtttttga cgccttctgt aatgcagcct gatcctcagt 540 ggttaaattt tcaaatcctg ggagtccctt ggtaaacttc attagccctt gtatgtgcag 600 gactgatacc tctgagagtc tcagaaaact tagttcgggg ttggaaccct cctgcagaag 660 tttgcttgtt tctcccaagg gaatcatgga tttttggtga gcagtcacta tggtattcag 720 aagccgatgt tcctcttgag ttagagtcat gttgtcctga acccctttcc cagatctggt 780 gctggatgac acgtgtttgg tgtctgctcc ttcatcttcc acttggatgg cagggtacag 840 Seite 40 L-0020-Ol-WO-O1.ST25 ggcaggcccg tgcttgaagt tcttgcgaag tctctttgac ttacactgga tctctgtgag 900 caaacattct gccaacatcc ccaccgcctt acacttcttc agtctgcact cttggcattt 960 tctgcgcatg tacatgtcca tttcacagtg accaccgttc ttgcaagaat acactgcatt 1020 cttggtaatg ctacgccgga aaaagccttt gcacccctca caagtaagtg cattgtagtg 1080 atatcctgat gccttatcac cgcagaccat gcagagcatg tcatcttgac ccttccagcg 1140 taaagaacaa gttgctctgc acctttgtgc cccagggaat tcatcttcat catcgtgaac 1200 .
aaccacataa gtggactgac cagattctcg agcgtcgagt ccacaccaac tgcctccact 1260 gta.Gt9tg9a.. t cGa~.gceat.- gtgtgttc~aa ~~tgacattgt aa~gavg~fJg ac'~Lgt~at~gc~ . 1:j20.: . . ..
tggaggaaac tgagcggtag aatattgaca atagggtggt tcttggaaat ctgtatcaca 1380 cagctgataa tggaattgtt ctggcaaaat atctggtttt attaacat 1428 <210>24 <211>475 <212>PRT

<213>Mus musculus <400> 24 Met Leu Ile Lys Pro Asp Ile Leu Pro Glu Gln Phe His Tyr Gln Leu Cys Asp Thr Asp Phe Gln Glu Pro Pro Tyr Cys Gln Tyr Ser Thr Ala Gln Phe Pro Pro Ala Leu Gln Ser Pro Ser Leu Gln Ser His Phe Asn Thr His Gly Leu Asp Pro Gln Tyr Ser Gly Gly Ser Trp Cys Gly Leu Asp Ala Arg Glu Ser Gly Gln Ser Thr Tyr Val Val Val His Asp Asp Glu Asp Glu Phe Pro Gly Ala Gln Arg Cys Arg Ala Thr Cys Ser Leu Seite 41 L-0020-Ol-WO-Ol.ST25 Arg Trp Lys Gly Gln Asp Asp Met Leu Cys Met Ual Cys Gly Asp Lys Ala Ser Gly Tyr His Tyr Asn Ala Leu Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Ile Thr Lys Asn Ala Ual Tyr Ser Cys Lys Asn Gly Gly His Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Cys Lys Ala Ual Gly Met Leu Ala Glu Cys Leu Lei. ihr~ Glu ~tle::Gl;s. Cys.-Lys,:Se~-r.:i ys~A.r~:c~ I_e~.t ArYgr.Lys.~A~n phe_..~4.y~ ..- .. . .... : : ~ _ ~ ..~

His Gly Pro Ala Leu Tyr Pro Ala Ile Gln Val Glu Asp Glu Gly Ala 195 200 ~ 205 Asp Thr Lys His Ual Ser Ser Ser Thr Arg Ser Gly Lys Gly Ual Gln Asp Asn Met Thr Leu Thr Gln Glu Glu His Arg Leu Leu Asn Thr Ile Ual Thr Ala His Gln Lys Ser Met Ile Pro Leu Gly Glu Thr Ser Lys Leu Leu Gln Glu Gly Ser Asn Pro Glu Leu Ser Phe Leu Arg Leu Ser Glu Ual Ser Ual Leu His Ile Gln Gly Leu Met Lys Phe Thr Lys Gly 275 280 ~ 285 Leu Pro Gly Phe Glu Asn Leu Thr Thr Glu Asp Gln Ala Ala Leu Gln Lys Ala Ser Lys Thr Glu Ual Met Phe Leu His Ual Ala Gln Leu Tyr Gly Gly Lys Asp Ser Thr Ser Gly Ser Thr Met Arg Pro Ala Lys Pro Ser Ala Gly Thr Leu Glu Ual His Asn Pro Ser Ala Asp Glu Ser Ual Seite 42 L-0020-Ol-WO-01.ST25 His Ser Pro Glu Asn Phe Leu Lys Glu Gly Tyr Pro Ser Ala Pro Leu Thr Gly Arg Met Ser Glu Leu His Ual Ser Asp Thr Glu Tyr Ala Leu Leu Thr Ala Thr Thr Ual Leu Phe Ser Asp Arg Pro Cys Leu Lys Asn Lys Gln His Ile Glu Asn Leu Gln Glu Pro Ual Leu Gln Leu Leu Phe Lys Phe Ser Lys Met Tyr His Pro Glu Asp Pro Gln Nis Phe Ala His Leu Il~e~ Gly Arg Leu Thr Glu Leu Arg Thr Leu Ser~His Ser His Ser Glu Ile Leu Arg Met Trp Lys Thr Lys Asp Pro Arg Leu Val Met Leu 450 455 . 460 Phe Ser Glu Lys Trp Asp Leu His Ser Phe Ser Seite 43

Claims (21)

CLAIMS:

1. A nucleic acid molecule coding for a nuclear receptor which is selected from the group comprising:
a) the nucleotide sequences set forth in SEQ ID NOs: 1, 4, 6, 8, 10, 12, 17, and/or 22;
b) or complement thereof as set forth in SEQ ID NOs; 2, 5, 7, 9, 11, 13, 18, and/or 23;
c) a nucleic acid which hybridizes to a nucleic acid having a nucleotide sequence which is the complement of the nucleotide sequence of SEQ ID NOs: 1, 4, 6, 8, 10, 12, 17, 20 and/or 22 under conditions of high stringency, and d) a nucleic acid which hybridizes to a nucleic acid having a nucleotide sequence which is the complement of the nucleotide sequence of SEQ ID NOs: 2, 5, 7, 9, 11, 13, 18, 21 and/or 23 under conditions of high stringency.

2. The nucleic acid molecule of claim 1 which is genomic DNA.

3. The nucleic acid molecule of claim 1 which is cDNA.

4. The nucleic acid molecule of claim 1 which is RNA.

5. A nucleic acid molecule comprising the. nucleic acid molecule of any of claims 1 to 4 and a label attached thereto.

6. A vector comprising the nucleic acid molecule of claim 1.

7. The vector of claim 6, which is an expression vector.

8. A host cell transfected with the vector of claim 6 or 7.

9. A host cell transfected with the expression vector of claim 7.

10. A method of producing a polypeptide comprising the step of culturing the host cell of claim 9 in an appropriate culture medium to, thereby, produce the polypeptide.

11.An isolated polypeptide encoded by any portion of the nucleic acid of claim 1.

12.A polypeptide selected from the group comprising:
the amino acid sequences set forth in SEQ ID NO: 3, 24 and/or 19.

13.A method for screening for agents which are capable of inhibiting the cellular function of the nuclear receptor L66 comprising the steps of:
a) contacting one or more candidate agents with a polypeptide according to claim 11 or 12 b) removing unbound agent(s) c) detecting whether the agent(s) interact with the polypeptide of the nuclear receptor.

14.A method for inhibiting the cellular function of the nuclear receptor L66, comprising the steps of:
a) contacting a cell with a binding agent of a polypeptide according to claim 11 or 12, whereby the cellular function of L66 is inhibited.

15. Method according to claim 14, characterized in that the binding agent is an antibody.

16. Method according to claim 14, characterized in that the binding agent is RNA.

17. Method according to claim 14, characterized in that the binding agent is an anti-sense oligonucleotide.

18. Method according to claim 14, characterized in that the binding agent is a ribozyme.

19. Method according to claim 14, characterized in that the cell is in a body.

20. Use of a nucleic acid or protein sequence according to SEQ ID NO.: 1, 4, 6, 8, 10, 12, 17, 20, 22, 2, 5, 7, 9, 11, 13, 18, 21, 3, 19, 24 and/or 23 for the construction of multiple nuclear receptor specific sequence alignments.

21. Use of the sequences according to claim 20 for the construction of protein sequence alignments.